Flavor compositions and pet food products containing the same

ABSTRACT

A flavor composition comprising at least one nucleotide derivative and/or at least one transmembrane compound that modulates, increases and/or enhances the activity of an umami receptor is provided that can be used to enhance the umami taste and/or palatability of food products. In certain, non-limiting embodiments, the flavor composition comprises at least one nucleotide derivative and/or at least one transmembrane compound, optionally at least one nucleotide, optionally at least one first amino acid, optionally at least one second amino acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage patent application under 35 U.S.C. § 371 of International Application No. PCT/US2015/065046, filed on Dec. 10, 2015, which claims priority to U.S. Provisional Application Ser. No. 62/090,138, filed on Dec. 10, 2014, the contents of each of which are incorporated by reference in their entireties, and to which priority is claimed.

FIELD

The presently disclosed subject matter relates to compounds that modulate the activity of an umami taste receptor, and to flavor compositions that include at least one such compound, as well as to methods of identifying such compounds. The flavor compositions can be used to enhance or modify the palatability, taste and/or flavor of pet food products. The flavor compositions can include combinations of compounds, and can be added to pet food products in various delivery system formats. The compounds that modulate the activity of an umami taste receptor can include one or more transmembrane compound, nucleotide derivative, nucleotide, first amino acid, second amino acid, or combinations thereof.

BACKGROUND

Taste profiles for edible compositions include basic tastes such as sweet, salt, bitter, sour, umami and kokumi. Taste profiles have also been described as including free fatty acid tastes. Chemical compounds that elicit these tastes are often referred to as tastants. It is hypothesized that tastants are sensed by taste receptors in the mouth and throat which transmit signals to the brain where the tastants and resulting taste profiles are registered. Taste receptors include the T1R class of taste receptors such as T1R1, T1R2 and T1R3, which interact as heterodimers to function as taste receptors. For example, T1R2/T1R3 responds to sweet stimuli and the T1R1/T1R3 heterodimer recognizes the umami taste. Cats and members of the Felidae family fail to express a functional T1R2 monomer indicating that the primary functional T1R class taste receptor in cats is the umami receptor, T1R1/T1R3. Further, cats have shown a preference for food compositions with an umami taste.

There are several notable differences between human T1R1/T1R3 and cat T1R1/T1R3 receptors. For example, human T1R1/T1R3 responds to the amino acid glutamate as an agonist, and to nucleotides, notably IMP and GMP, as positive allosteric modulators. Human T1R1/T1R3 also responds to compounds that bind the transmembrane domain of the receptor. For example, N-(heptan-4-yl)benzo[d][1,3] dioxole-5-carboxamide), which is a positive allosteric modulator of the receptor, was shown to bind to the human T1R1 transmembrane domain by domain swapping experiments (Zhang et. al. Proc Natl Acad Sci USA. 105(52):20930-4, 2008). Allosteric modulators that bind to the transmembrane domain are also known to modulate the activity of multiple members of the Class III GPCRs, including mGluR receptors and the CaSR receptor. However, both human and cat T1R1/T1R3 respond synergistically to combinations of nucleotides and amino acids.

Pet food manufacturers have a long-standing desire to provide pet food products that have high nutritional value. In addition, and with particular regard to cat and dog foods, pet food manufacturers desire a high degree of palatability so that pets can receive the full nutritional benefit from their food. Domestic animals, especially cats, are notoriously fickle in their food preferences, and often refuse to eat a pet food product that it has accepted over some time or refuse to eat any more than a minimal amount of a pet food product. As a result, pet owners frequently change types and brands of pet food in order to maintain their pets in a healthy and contented condition.

While there have been recent advances in taste and flavor technologies, there remains a need for compounds that can enhance or modify the palatability of pet food products by enhancing or modifying the taste, texture and/or flavor profiles of the pet food product. The enhancement or modification can be to increase the intensity of a desirable attribute, to replace a desirable attribute that is not present or somehow lost in the pet food product, or to decrease the intensity of an undesirable attribute. In particular, it is desirable to increase the intensity of a tastant in a pet food product. Therefore, there remains a need in the art for compositions to enhance the palatability and/or umami taste of pet food products.

SUMMARY OF THE INVENTION

The presently disclosed subject matter is directed to flavor compositions and methods for making and modifying such compositions across a variety of pet food products. Specifically, the present disclosure is directed to compositions comprising one or more transmembrane compounds and/or nucleotide derivatives that enhance, increase and/or modulate the activity of the umami receptor, T1R1/T1R3.

In certain embodiments of the present disclosure, the flavor composition comprises a nucleotide derivative of Formula Nt-1:

wherein X, R₁, R₂, R₄, R₅, R₁₄ and R₁₅ are described herein below. The present disclosure provides salts and stereoisomers of the compounds of Formula Nt-1.

In certain embodiments of the present disclosure, the flavor composition comprises one or more nucleotide derivatives of Formulas Nt-2 to Nt-55, as described herein.

In certain embodiments of the present disclosure, the flavor composition further comprises one or more transmembrane compounds of Formulas Tm-1 to Tm-104, as described herein.

In certain embodiments, the flavor composition further comprises one or more first amino acids and/or one or more second amino acids. In certain embodiments, the flavor composition further comprises at least one, two, three, four, five or more first amino acids and/or at least one, two, three, four, five or more second amino acids. In certain embodiments, the flavor composition further comprises at least one first amino acid and/or at least one second amino acid. In certain embodiments, the flavor composition comprises at least two first amino acids and/or at least one second amino acid. In certain embodiments, the flavor composition further comprises at least one first amino acid and/or at least two second amino acids. In certain embodiments, the flavor composition further comprises at least two first amino acids and/or at least two second amino acids.

Non-limiting examples of a first amino acid include tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine, and combinations thereof.

Non-limiting examples of a second amino acid include asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxyl proline, arginine, cystine, glutamine, lysine, valine, omithine, monosodium glutamate, taurine, and combinations thereof.

In certain embodiments, the flavor composition further comprises alanine.

In certain embodiments, the flavor composition further comprises glycine.

In certain embodiments, the flavor composition further comprises histidine.

In certain embodiments, the flavor composition can further comprise at least one nucleotide or nucleotide derivative. Non-limiting examples of nucleotides include guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), inosine monophosphate (IMP), inosine diphosphate (IDP), inosine triphosphate (ITP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), xanthosine monophosphate (XMP), xanthosine diphosphate (XDP), xanthosine triphosphate (XTP), and combinations thereof.

In one embodiment, the flavor composition can further comprise a nucleotide selected from the group consisting of guanosine monophosphate (GMP), inosine monophosphate (IMP), and a combination thereof.

In other embodiments, the flavor composition further comprises alanine and guanosine monophosphate (GMP).

In other embodiments, the flavor composition further comprises glycine and guanosine monophosphate (GMP).

In other embodiments, the flavor composition further comprises histidine and guanosine monophosphate (GMP).

In yet other embodiments, the flavor composition further comprises alanine and inosine monophosphate (IMP).

In yet other embodiments, the flavor composition further comprises glycine and inosine monophosphate (IMP).

In yet other embodiments, the flavor composition further comprises histidine and inosine monophosphate (IMP).

In other embodiments, the flavor composition further comprises proline and an amino acid selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.

In other embodiments, the flavor composition further comprises threonine and an amino acid selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.

In certain embodiments, the flavor composition further comprises glutamic acid and an amino acid selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.

In certain embodiments, the present disclosure provides for a food product comprising a flavor composition as described herein, wherein the flavor composition is present at a concentration of about 0.0001 weight % to about 10 weight % of the food product. In other embodiments, the flavor composition is present at a concentration of from about 0.001 ppm to about 1,000 ppm of the food product. In yet other embodiments, the flavor composition is present at a concentration of from about 1 pM to about 1 M, or about 1 μM to about 1 M of the food product.

In certain embodiments, the flavor composition is present in an amount effective to increase the palatability of the food product, as determined by a panel of taste testers.

In other embodiments, the present disclosure provides for a method of increasing an umami taste intensity in a food product comprising admixing a food product with the flavor compositions described herein, wherein the flavor composition is present at a concentration of from about 0.0001 weight % to about 10 weight % of the admixture. In other embodiments, the flavor composition is present at a concentration of from about 0.001 ppm to about 1,000 ppm of the admixture. In yet other embodiments, the flavor composition is present at a concentration of from about 1 pM to about 1 M, or about 1 μM to about 1 M of the admixture.

In other embodiments, the present disclosure provides for a method of increasing an umami taste intensity in a food product comprising admixing a food product with the flavor compositions described herein, wherein the flavor composition is present in an amount effective to increase the palatability of the food product, as determined by a panel of taste testers. In certain embodiments, the increase in umami taste intensity comprises an increase in umami aftertaste.

The present disclosure also provides for methods of preparing a food product comprising a flavor composition described herein, wherein the method comprises thermal processing of a food product precursor, wherein the flavor composition is generated during the thermal processing. Examples of thermal processing include, for example, sterilization, retorting, extrusion, injection molding or combinations thereof.

In certain embodiments, the food products described herein comprise pet food products, for example, feline pet food products such as wet and/or dry feline pet food products. In other examples, the pet food products comprise canine pet food products such as wet and/or dry canine pet food products.

In other embodiments, the food products described herein comprise human food products.

The foregoing has outlined rather broadly the features and technical advantages of the present application in order that the detailed description that follows may be better understood. Additional features and advantages of the application will be described hereinafter which form the subject of the claims of the application. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present application. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the application as set forth in the appended claims. The novel features which are believed to be characteristic of the application, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dose response curves for guanosine monophosphate (GMP) as a test compound for activating the cat T1R1/T1R3 umami receptor in vitro. In the present figure (as well as in FIGS. 2-12, 14, and 38-45, four curves are shown: the test compound in buffer, the test compound in buffer with 20 mM alanine, the test compound in buffer with 0.2 mM IMP, and the test compound in 20 mM alanine and 0.2 mM IMP. Horizontal axes are test compound concentrations in mM. Vertical axes are receptor responses measured as ΔF/F in a fluorescent assay or as Lum in a luminescence assay.

FIG. 2 shows dose response curves for 2′-deoxyadenosine-3′, 5′-O-bisphosphate.

FIG. 3 shows dose response curves for inosine 5′-diphosphate (IDP) sodium salt.

FIG. 4 shows dose response curves for 2′-/3′-O-(2-aminoethylcarbamoyl) adenosine-5′ O-monophosphate.

FIG. 5 shows dose response curves for 2′-/3′-O-(2-aminoethylcarbamoyl) guanosine-5′-O-monophosphate.

FIG. 6 shows dose response curves for inosine triphosphate (ITP) trisodium salt.

FIG. 7 shows dose response curves for N6-benzoyladenosine-5′-O-monophosphate.

FIG. 8 shows dose response curves for adenosine 5′-O-thiomonophosphate dilithium salt.

FIG. 9 shows dose response curves for adenosine 3′, 5′ diphosphate sodium salt and alanine (1:1000).

FIG. 10 shows dose response curves for adenosine 3′, 5′ diphosphate sodium salt and alanine (1:100).

FIG. 11 shows dose response curves for adenosine 3′, 5′ diphosphate sodium salt and alanine (1:10).

FIG. 12 shows dose response curves for adenosine 3′, 5′ diphosphate sodium salt.

FIG. 13 shows dose response curves for inosine monophosphate (IMP) for activating cat T1R1/T1R3. IMP was used as a control for the experiments described by FIGS. 1-13 and 38-45.

FIG. 14 shows an overall structural model of the VFT domain of cat T1R1/T1R3 bound to IMP and L-Alanine. L-Alanine and IMP are shown bound in the active site between the upper and lower globes of the VFT domain.

FIG. 15 shows a model of the cat T1R1 VFT bound to L-Alanine. L-Alanine binds to a hinge region of the VFT domain. Putative hydrogen bond, salt-bridge, and Pi-cation interactions are shown between L-Alanine and the following T1R1 amino acids: Thr149, Ser172, Tyr220, Thr148, Glu170, and Asp302. The interactions are shown as dotted lines. Glu170, and Asp302 coordinate the zwitterionic nitrogen of bound amino acids while electrostatically disfavoring binding of L-Glutamate and L-aspartic acid, the native ligands for the human umami receptor.

FIG. 16 shows an in silico model of the cat T1R1 VFT bound to GMP. Putative hydrogen bond and salt bridge interactions are shown as dotted lines between the phosphate of GMP and His47, His71, Arg277, and Asn69; the sugar of GMP and Asp302 and Ser306; and the GMP base and Ser384, His308, and Ala380.

FIG. 17 shows an in silico model of the cat T1R1 VFT that shows that Asp302 of T1R1 may simultaneously coordinate the zwitterionic backbone nitrogen of a bound amino acid (L-alanine to the left) and the sugar molecule of a bound nucleotide (GMP to the right).

FIG. 18 shows an in silico model of the cat T1R1 VFT that shows putative interactions between T1R1 and a bound 3′5′ diphosphate.

FIG. 19 shows an in silico model of the cat T1R1 VFT that shows putative interactions between T1R1 and a bound XMP.

FIG. 20 shows an in silico model of the cat T1R1 VFT that shows putative interactions between T1R1 and a bound N-Acetyl-5′-GMP.

FIG. 21 shows an in silico model of the cat T1R1 VFT that shows putative interactions between T1R1 and a bound 2′-3′ AEC-5′-AMP.

FIG. 22 shows an in silico model of the cat T1R1 VFT that shows putative interactions between T1R1 and a bound 5′d-GMPS.

FIG. 23 shows an in silico model of the cat T1R1 VFT that shows putative interactions between T1R1 and a bound 5′-O-2-thiodiphospate.

FIG. 24 shows an in silico model of the cat T1R1 VFT that shows putative interactions between T1R1 and a bound 6-Thioguanosine-5′-O-monophosphate.

FIG. 25 shows an in silico model of the cat T1R1 VFT that shows putative interactions between T1R1 and a bound CMP.

FIG. 26 shows an in silico model of the cat T1R1 VFT bound to adenosine 3′5′ diphosphate. Putative hydrogen bond and salt bridge interactions are shown as dotted lines between the phosphates of Adenosine 3′5′ diphosphate and His71, His47, Asn69, Arg281, Arg277, His308, and Ile309; the sugar of Adenosine 3′5′ diphosphate and Asp302, and Ser306; and the Adenosine 3′5′ diphosphate base and Ser384.

FIG. 27 show an in silico model of the cat T1R1 transmembrane domain. A transmembrane compound N-benzyl-L-phenylalanine methyl ester is shown docked within the transmembrane region of T1R1.

FIG. 28 shows an in silico model of N-benzyl-L-phenylalanine methyl ester docked within the transmembrane region of T1R1.

FIG. 29 shows an in silico model of 2-amino-N-phenethylbenzamide docked within the transmembrane region of T1R1.

FIG. 30 shows an in silico model of N-(2-(1H-indol-3-yl)ethyl)nicotinamide docked within the transmembrane region of T1R1.

FIG. 31 shows an in silico model of 1-benzyl-3-(2-(5-chlorothiophen-2-yl)-2-oxoethyl)imidazolidine-2,4,5-trione docked within the transmembrane region of T1R1.

FIG. 32 shows an in silico model of ethyl (2,2-diphenylacetyl)carbamate docked within the transmembrane region of T1R1.

FIG. 33 shows an in silico model of 2-((3,5-dichlorophenyl)carbamoyl) cyclohexanecarboxylic acid docked within the transmembrane region of T1R1.

FIG. 34 shows an in silico model of N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide docked within the transmembrane region of T1R1.

FIG. 35 shows in silico modeling of 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione docked within the transmembrane region of T1R1.

FIG. 36 shows in silico modeling of a 1H-imidazo[4,5-c]pyridin-2(3H)-one derivative compound docked within the transmembrane region of T1R1.

FIG. 37 shows dose response curves for 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione.

FIG. 38 shows dose response curves for 1-(2-bromophenyl)-3-((1R, 2S)-2-hydroxy-2,3-dihydro-1H-inden-1-yl)urea.

FIG. 39 shows dose response curves for N-(benzo[d][1,3]dioxol-5-yl)-2-propylpentanamide.

FIG. 40 shows dose response curves for N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide.

FIG. 41 shows dose response curves for N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide.

FIG. 42 shows dose response curves for (E)-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide.

FIG. 43 shows dose response curves for 2-((5-(4-(methylthio)phenyl)-2H-tetrazol-2-yl)methyl)pyridine.

FIG. 44 shows dose response curves for N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide in the presence of GMP and phenylalanine.

FIG. 45 shows a graph plotting the ΔF/F₀ values for N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide in the presence of GMP and phenylalanine.

FIG. 46 shows dose response curves for positive and negative controls in activating cat T1R1/T1R3 for the experiments described by FIGS. 1-14 and 38-45. Dose response curves for amino acids were determined in the presence of 0.2 mM IMP. Dose response curves for nucleotides were determined in the presence of 20 mM alanine.

FIG. 47 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 48 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 49 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 50 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 51 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 52 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 53 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 54 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 55 shows in silico modeling of a transmembrane compound, according to one exemplary embodiment of the disclosed subject matter, docked within the transmembrane region of T1R1.

FIG. 56A-B shows (A) the activation of T1R1/T1R3 by 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 57A-B shows (A) the activation of T1R1/T1R3 by 4-benzyl-3-butyl-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-1H-1,2,4-triazol-5(4H)-one in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by 4-benzyl-3-butyl-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-1H-1,2,4-triazol-5(4H)-one alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 58A-B shows (A) the activation of T1R1/T1R3 by 2-((3,5-dichlorophenyl)carbamoyl)cyclohexanecarboxylic acid in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by 2-((3,5-dichlorophenyl)carbamoyl)cyclohexanecarboxylic acid alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 59A-B shows (A) the activation of T1R1/T1R3 by 4-acetamido-N-(1-(2-hydroxyethyl)-3-phenyl-1H-pyrazol-5-yl)benzamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by 4-acetamido-N-(1-(2-hydroxyethyl)-3-phenyl-1H-pyrazol-5-yl)benzamide alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 60A-B shows (A) the activation of T1R1/T1R3 by (Diphenylacetyl)-carbamic acid ethyl ester in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by (Diphenylacetyl)-carbamic acid ethyl ester alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 61A-B shows (A) the activation of T1R1/T1R3 by N,N′-(butane-1,4-diyl)dinicotinamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by N,N′-(butane-1,4-diyl)dinicotinamide alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 62A-B shows (A) the activation of T1R1/T1R3 by N-phenethylnicotinamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by N-phenethylnicotinamide alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 63A-B shows (A) the activation of T1R1/T1R3 by 2-amino-N-phenethylbenzamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by 2-amino-N-phenethylbenzamide alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 64A-B shows (A) the activation of T1R1/T1R3 by N-phenethylbenzo[d][1,3]dioxole-5-carboxamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by N-phenethylbenzo[d][1,3]dioxole-5-carboxamide alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 65A-B shows (A) the activation of T1R1/T1R3 by N-phenethylbenzamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by N-phenethylbenzamide alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 66A-B shows (A) the activation of T1R1/T1R3 by N-Benzoyl-DL-Leucinamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by N-Benzoyl-DL-Leucinamide alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 67A-B shows (A) the activation of T1R1/T1R3 by N-(2-(1H-Indol-3-yl)ethyl)nicotinamide in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by N-(2-(1H-Indol-3-yl)ethyl)nicotinamide alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 68A-B shows (A) the activation of T1R1/T1R3 by N-benzyl-L-phenylalanine methyl ester hydrochloride in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by N-benzyl-L-phenylalanine methyl ester hydrochloride alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 69A-B shows (A) the activation of T1R1/T1R3 by 6-Thioguanosine-5′-O-diphosphate (6-T-GDP) in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by 6-Thioguanosine-5′-O-diphosphate (6-T-GDP) alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 70A-B shows (A) the activation of T1R1/T1R3 by 6-Chloropurine riboside-5′-O-triphosphate (6-Cl-PuTP) in the presence of 20 mM L-alanine or 0.2 mM IMP, and (B) dose response curves for activating T1R1/T1R3 by 6-Chloropurine riboside-5′-O-triphosphate (6-Cl-PuTP) alone in buffer, or in the presence of 20 mM L-alanine, 0.2 mM IMP, or both 20 mM L-alanine and 0.2 mM IMP.

FIG. 71 shows dose response curves for positive and negative controls in activating cat T1R1/T1R3 for the experiments described by FIGS. 57-75. Dose response curves for amino acids were determined in the presence of 0.2 mM IMP. Dose response curves for nucleotides were determined in the presence of 20 mM alanine.

FIG. 72A-D shows the activation of T1R1/T1R3: (A) by the transmembrane compound 1-benzyl-3-(2-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7-yl)-2-oxoethyl)imidazolidine-2,4,5-trione alone (agonist profile assessment), and (B) and (C) in the presence of GMP and Alanine (PAM profile assessment). (D) shows a graphical representation of data from both (A) and (B). The transmembrane compound exhibited PAM activity.

FIG. 73A-F shows the activation of T1R1/T1R3: (A) by the transmembrane compound 1-benzyl-3-(2-(5-chlorothiophen-2-yl)-2-oxoethyl)imidazolidine-2,4,5-trione alone (agonist profile assessment), and (B) and (C) in the presence of GMP and Alanine (PAM profile assessment). (D) shows a graphical representation of data from both (A) and (B). (E) and (F) show dose response curves for the transmembrane compound as GMP concentration was held constant and Ala concentration was varied, and when Ala concentration was held constant and GMP concentration was varied in cells expressing T1R1/T1R3 (induced) and mock control cells that did not express T1R1 (uninduced). The transmembrane compound exhibited PAM activity.

FIG. 74A-E shows the activation of T1R1/T1R3: (A) by the transmembrane compound N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide alone (agonist profile assessment), and (B) in the presence of GMP and Alanine (PAM profile assessment). (C) shows a graphical representation of data from both (A) and (B). (D) and (E) show dose response curves for the transmembrane compound as GMP concentration was held constant and Ala concentration was varied, and when Ala concentration was held constant and GMP concentration was varied in cells expressing T1R1/T1R3 (induced, (D)) and mock control cells that did not express T1R1 (uninduced, (E)). The transmembrane compound exhibited agonist and PAM activity.

FIG. 75A-E shows the activation of T1R1/T1R3: (A) by the transmembrane compound N-benzyl-L-phenylalanine methyl ester hydrochloride alone (agonist profile assessment), and (B) in the presence of GMP and Alanine (PAM profile assessment). (C) shows a graphical representation of data from both (A) and (B). (D) and (E) show dose response curves for the transmembrane compound as GMP concentration was held constant and Ala concentration was varied, and when Ala concentration was held constant and GMP concentration was varied in cells expressing T1R1/T1R3 (induced, (D)) and mock control cells that did not express T1R1 (uninduced, (E)). The transmembrane compound exhibited agonist and PAM activity.

FIG. 76A-E shows the activation of T1R1/T1R3: (A) by the transmembrane compound N-Benzyl-D-Phenylalanine methyl ester hydrochloride alone (agonist profile assessment), and (B) in the presence of GMP and Alanine (PAM profile assessment). (C) shows a graphical representation of data from both (A) and (B). (D) and (E) show dose response curves for the transmembrane compound as GMP concentration was held constant and Ala concentration was varied, and when Ala concentration was held constant and GMP concentration was varied in cells expressing T1R1/T1R3 (induced, (D)) and mock control cells that did not express T1R1 (uninduced, (E)). The transmembrane compound exhibited agonist and PAM activity.

FIG. 77A-E shows the activation of T1R1/T1R3: (A) by the transmembrane compound Benzyl-L-leucine methyl ester hydrochloride alone (agonist profile assessment), and (B) in the presence of GMP and Alanine (PAM profile assessment). (C) shows a graphical representation of data from both (A) and (B). (C) and (E) show dose response curves for the transmembrane compound as GMP concentration was held constant and Ala concentration was varied, and when Ala concentration was held constant and GMP concentration was varied in cells expressing T1R1/T1R3 (induced, (D)) and mock control cells that did not express T1R1 (uninduced, (E)). The transmembrane compound exhibited agonist and PAM activity.

FIG. 78A-E shows the activation of T1R1/T1R3: (A) by the transmembrane compound Methyl-2-benzylamino-2-phenylacetate alone (agonist profile assessment), and (B) in the presence of GMP and Alanine (PAM profile assessment). (C) shows a graphical representation of data from both (A) and (B). (D) and (E) show dose response curves for the transmembrane compound as GMP concentration was held constant and Ala concentration was varied, and when Ala concentration was held constant and GMP concentration was varied in cells expressing T1R1/T1R3 (induced, (D)) and mock control cells that did not express T1R1 (uninduced, (E)). The transmembrane compound exhibited agonist and PAM activity.

FIG. 79A-E shows the activation of T1R1/T1R3: (A) by the transmembrane compound L-Phenylalanine benzyl ester hydrochloride alone (agonist profile assessment), and (B) in the presence of GMP and Alanine (PAM profile assessment). (C) shows a graphical representation of data from both (A) and (B). (D) and (E) show dose response curves for the transmembrane compound as GMP concentration was held constant and Ala concentration was varied, and when Ala concentration was held constant and GMP concentration was varied in cells expressing T1R1/T1R3 (induced, (D)) and mock control cells that did not express T1R1 (uninduced, (E)). The transmembrane compound exhibited agonist and PAM activity.

FIG. 80A-B shows the activation of T1R1/T1R3: (A) by the transmembrane compound 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione alone (agonist profile assessment), and (B) in the presence of GMP and Alanine (PAM profile assessment). The transmembrane compound exhibited agonist activity.

FIG. 81 shows a cat T1R1 receptor nucleic acid sequence (SEQ ID NO: 1).

FIG. 82 shows a cat T1R1 receptor amino acid sequence (SEQ ID NO:2).

FIG. 83 shows a cat T1R3 receptor nucleic acid sequence (SEQ ID NO:3).

FIG. 84 shows a cat T1R3 receptor amino acid sequence (SEQ ID NO:4).

DETAILED DESCRIPTION

To date, there remains a need for a flavor modifier that can provide a desired level of umami flavor to increase and/or enhance the palatability of various cat pet food products. The present application relates to flavor compositions that include at least one nucleotide derivative and/or transmembrane compound. The flavor compositions can be used to increase the palatability and/or enhance or modify the taste of various pet food products such as a nutritionally-complete pet food. In certain embodiments, the flavor compositions can be used to increase the umami taste of a pet food product. The flavor compositions can further include combinations of compounds, including nucleotides and/or amino acids, and can be added to pet food products in various delivery system formats.

1. DEFINITIONS

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.

As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Still further, the terms “having,” “including,” “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

As used herein, “taste” refers to a sensation caused by activation or inhibition of receptor cells in a subject's oral cavity. In certain embodiments, taste can be selected from the group consisting of sweet, sour, salt, bitter, kokumi and umami. In certain embodiments, “taste” can include free fatty acid taste. See, e.g., Cartoni et al., J. of Neuroscience, 30(25): 8376-8382 (2010), the contents of which are incorporated herein by reference. In certain embodiments, a taste is elicited in a subject by a “tastant.” In certain embodiments, a tastant is a synthetic tastant. In certain embodiments, the tastant is prepared from a natural source.

As used herein, “taste profile” refers to a combination of tastes, such as, for example, one or more of a sweet, sour, salt, bitter, umami, kokumi and free fatty acid taste. In certain embodiments, a taste profile is produced by one or more tastant that is present in a composition at the same or different concentrations. In certain embodiments, a taste profile refers to the intensity of a taste or combination of tastes, for example, a sweet, sour, salt, bitter, umami, kokumi and free fatty acid taste, as detected by a subject or any assay known in the art. In certain embodiments, modifying, changing or varying the combination of tastants in a taste profile can change the sensory experience of a subject.

In certain embodiments, “aftertaste” refers to the taste intensity of a food product that is perceived after the food product is removed from the mouth or oral cavity.

As used herein, “flavor” refers to one or more sensory stimuli, such as, for example, one or more of taste (gustatory), smell (olfactory), touch (tactile) and temperature (thermal) stimuli. In certain non-limiting embodiments, the sensory experience of a subject exposed to a flavor can be classified as a characteristic experience for the particular flavor. For example, a flavor can be identified by the subject as being, but not limited to, a floral, citrus, berry, nutty, caramel, chocolate, peppery, smoky, cheesy, meaty, etc., flavor. As used herein, a flavor composition can be selected from a liquid, solution, dry powder, spray, paste, suspension and any combination thereof. The flavor can be a natural composition, an artificial composition, a nature identical, or any combination thereof.

As used interchangeably herein, “aroma” and “smell” refer to an olfactory response to a stimulus. For example, and not by way of limitation, an aroma can be produced by aromatic substances that are perceived by the odor receptors of the olfactory system.

As used herein, “flavor profile” refers to a combination of sensory stimuli, for example, tastes, such as sweet, sour, bitter, salty, umami, kokumi and free fatty acid tastes, and/or olfactory, tactile and/or thermal stimuli. In certain embodiments, the flavor profile comprises one or more flavors which contribute to the sensory experience of a subject. In certain embodiments, modifying, changing or varying the combination of stimuli in a flavor profile can change the sensory experience of a subject.

As used herein “admixing,” for example, “admixing the flavor composition or combinations thereof of the present application with a food product,” refers to the process where the flavor composition, or individual components of the flavor composition, is mixed with or added to the completed product or mixed with some or all of the components of the product during product formation or some combination of these steps. When used in the context of admixing, the term “product” refers to the product or any of its components. This admixing step can include a process selected from the step of adding the flavor composition to the product, spraying the flavor composition on the product, coating the flavor composition on the product, suspending the product in the flavor composition, painting the flavor composition on the product, pasting the flavor composition on the product, encapsulating the product with the flavor composition, mixing the flavor composition with the product and any combination thereof. The flavor composition can be a solution, liquid, dry powder, spray, paste, suspension and any combination thereof.

In certain embodiments, the nucleotide derivatives and/or transmembrane compounds of a flavor composition can be generated during the thermal processing of a pet food product, e.g., sterilization, retorting, injection molding and/or extrusion, from precursor compounds present in the pet food product. In certain embodiments, a nucleotide derivative and/or transmembrane compound of a flavor composition can be generated during the processing of a pet food product and additional components of the flavor composition, e.g., a nucleotide and/or an amino acid, can be added to the pet food product by admixing.

As used herein, “ppm” means parts-per-million and is a weight relative parameter. A part-per-million is a microgram per gram, such that a component that is present at 10 ppm is present at 10 micrograms of the specific component per 1 gram of the aggregate mixture.

As used herein, “palatability” can refer to the overall willingness of a human or non-human animal, for example, a companion animal, to eat a certain food product. Increasing the “palatability” of a food product can lead to an increase in the enjoyment and acceptance of the food by the human or non-human animal to ensure the human or non-human animal eats a “healthy amount” of the food. The term “healthy amount” of a food as used herein refers to an amount that enables the human or non-human animal to maintain or achieve an intake contributing to its overall general health in terms of micronutrients, macronutrients and calories, for example, such as set out in the “Mars Petcare Essential Nutrient Standards.” In certain embodiments, “palatability” can mean a relative preference of a human or non-human animal for one food product over another. For example, when a human or non-human animal shows a preference for one of two or more food products, the preferred food product is more “palatable,” and has “enhanced palatability.” In certain embodiments, the relative palatability of one food product compared to one or more other food products can be determined, for example, in side-by-side, free-choice comparisons, e.g., by relative consumption of the food products, or other appropriate measures of preference indicative of palatability. Palatability can be determined by a standard testing protocol in which the animal has equal access to both food products such as a test called “two-bowl test” or “versus test.” Such preference can arise from any of the animal's senses, but can be related to, inter alia, taste, aftertaste, smell, mouth feel and/or texture.

The term “pet food” or “pet food product” means a product or composition that is intended for consumption by a companion animal, such as cats, dogs, guinea pigs, rabbits, birds and horses. For example, but not by way of limitation, the companion animal can be a “domestic” cat such as Felis domesticus. In certain embodiments, the companion animal can be a “domestic” dog, e.g., Canis lupus familiaris. A “pet food” or “pet food product” includes any food, feed, snack, food supplement, liquid, beverage, treat, toy (chewable and/or consumable toys), meal substitute or meal replacement.

The term “human food” or “human food product” means a product or composition that is intended for consumption by a human. A “human food” or “human food product” includes any food, feed, snack, food supplement, liquid, beverage, treat, toy (chewable and/or consumable toys), meal substitute or meal replacement.

In certain embodiments, a “food product” includes human and/or pet food products.

As used herein “nutritionally-complete” refers to food product, for example, a pet food product, that contains all known required nutrients for the intended recipient of the food product, in appropriate amounts and proportions based, for example, on recommendations of recognized or competent authorities in the field of companion animal nutrition. Such foods are therefore capable of serving as a sole source of dietary intake to maintain life, without the addition of supplemental nutritional sources.

As used herein “flavor composition” refers to at least one compound or biologically acceptable salt thereof that modulates, including enhancing, multiplying, potentiating, decreasing, suppressing, or inducing, the tastes, smells, flavors and/or textures of a natural or synthetic tastant, flavoring agent, taste profile, flavor profile and/or texture profile in an animal or a human. In certain embodiments, the flavor composition comprises a combination of compounds or biologically acceptable salts thereof. In certain embodiments, the flavor composition includes one or more excipients.

As used herein “agonist” refers to at least one compound or biologically acceptable salt thereof that modulates, including enhancing, multiplying, potentiating, or inducing the activity of a receptor to which it binds or otherwise interacts with. In certain embodiments, the term is used to describe compounds that act alone to activate the receptor, or to describe “positive allosteric modulator” (also known as “PAM”) compounds, which positively enhance the action of other agonists.

As used herein, “synergy,” “synergistically” or “synergistic effect” refers to an effect produced by two or more individual components in which the total effect produced by these components, when utilized in combination, is greater than the sum of the individual effects of each component acting alone. As used herein, the term “synergistically effective” refers to any combined amount of a nucleotide derivative and/or transmembrane compound, and an additional compound (e.g., an amino acid, nucleotide, or a compound that binds to the transmembrane domains of T1R1 or T1R3 (see, for example, Zhang et al., Proc Natl Acad Sci USA. 2008 Dec. 30; 105(52):20930-4, Epub 2008 Dec. 22)), that exhibits synergistic activation of the T1R1/T1R3 receptor or increased palatability of a pet food product.

The term “alkyl” refers to a straight or branched C₁-C₂₀ (preferably C₁-C₆) hydrocarbon group consisting solely of carbon and hydrogen atoms, containing no unsaturation, and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl).

The term “alkenyl” refers to a C₂-C₂₀ (preferably C₂-C₁₂) aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be a straight or branched chain, e.g., ethenyl, 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl.

The term “alkynyl” refers to a C₂-C₂₀ (preferably C₂-C₁₂) aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and which may be a straight or branched chain, e.g., ethynyl, 1-propynyl, 2-propynyl.

The term “cycloalkyl” denotes an unsaturated, non-aromatic mono- or multicyclic hydrocarbon ring system (containing, for example, C₃-C₆) such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. Examples of multicyclic cycloalkyl groups (containing, for example, C₆-C₁₅) include perhydronapththyl, adamantyl and norbomyl groups bridged cyclic group or sprirobicyclic groups, e.g., spiro (4,4) non-2-yl.

The term “cycloalkalkyl” refers to a cycloalkyl as defined above directly attached to an alkyl group as defined above, that results in the creation of a stable structure such as cyclopropylmethyl, cyclobutylethyl, or cyclopentylethyl.

The term “ethereal” refers to an alkyl group or cycloalkyl group as defined above having at least one oxygen incorporated into the alkyl chain, e.g., methyl ethyl ether, diethyl ether, tetrahydrofuran. Such groups can also be described as alkoxyalkyl or alkoxycycloalkyl groups.

The term “aminoalkyl” refers to an alkyl group or a cycloalkyl group as defined above having at least one nitrogen atom, e.g., n-butyl amine and tetrahydrooxazine.

The term “aryl” refers to aromatic radicals having in the range of about 6 to about 14 carbon atoms such as phenyl, naphthyl, tetrahydronapthyl, indanyl, biphenyl.

The term “arylalkyl” refers to an aryl group as defined above directly bonded to an alkyl group as defined above, e.g., —CH₂C₆H₅, and —C₂H₄C₆H₅.

The term “heterocyclic” refers to a stable 3- to 15-membered ring radical, which consists of carbon atoms and one or more, for example, from one to five, heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this application, the heterocyclic ring radical may be a monocyclic or bicyclic ring system, which may include fused or bridged ring systems, and the nitrogen, carbon, oxygen or sulfur atoms in the heterocyclic ring radical may be optionally oxidized to various oxidation states. In addition, the nitrogen atom may be optionally quatemized; and the ring radical may be partially or fully saturated, or the ring radical may be fully unsaturated (i.e., heteroaromatic or heteroaryl aromatic).

The heterocyclic ring radical may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure.

The term “heteroaryl” refers to a heterocyclic ring wherein the ring is aromatic.

The term “heteroarylalkyl” refers to heteroaryl ring radical as defined above directly bonded to alkyl group. The heteroarylalkyl radical may be attached to the main structure at any carbon atom from alkyl group that results in the creation of a stable structure.

The term “heterocyclyl” refers to a heterocylic ring radical as defined above.

The heterocyclyl ring radical may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure.

In certain embodiments, the term “umami receptor” refers to a G protein coupled receptor (GPCR), for example, a T1R1/T1R3 GPCR. The umami receptor can be for example, a cat, dog, human or non-human mammal umami receptor.

In certain embodiments, the cat T1R1 is a protein comprising an amino acid sequence as set forth in SEQ ID NO:2, or a sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologous thereto (homology, as that term is used herein, may be measured using standard software such as BLAST or FASTA), and is encoded, for example, by a nucleic acid comprising a sequence as set forth in SEQ ID NO: 1, or a sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologous thereto (homology, as that term is used herein, may be measured using standard software such as BLAST or FASTA).

In certain embodiments, the cat T1R3 is a protein comprising an amino acid sequence as set forth in SEQ ID NO:4, or a sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologous thereto (homology, as that term is used herein, may be measured using standard software such as BLAST or FASTA), and is encoded, for example, by a nucleic acid comprising a sequence as set forth in SEQ ID NO:3, or a sequence at least 99, 98, 97, 96, 95, 90, 85 or 80 percent homologous thereto (homology, as that term is used herein, may be measured using standard software such as BLAST or FASTA).

2. NUCLEOTIDE DERIVATIVES

The present disclosure relates to flavor compositions that include at least one nucleotide derivative. In certain embodiments, the nucleotide derivative is an umami taste enhancing compound. The nucleotide derivatives disclosed herein were identified through the in silico modeling of the nucleotide derivatives within the binding pocket of the feline T1R1/T1R3 receptor (“Umami receptor”). The flavor compositions can be used to enhance or modify the palatability, taste or flavor of pet food product. The flavor compositions can include combinations of compounds, for example, combinations of one or more nucleotide derivatives and/or one or more amino acids and/or one or more nucleotides and/or one or more transmembrane compounds, as described herein, and can be added to pet food product compositions in various delivery system formats.

In certain embodiments, the nucleotide derivative can be a compound listed in Tables 2 and 5-13 below.

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-1 having the following structure:

wherein R₁ is selected from the group consisting of a substituted or unsubstituted purine or substituted or unsubstituted pyrimidine;

wherein R₂ is selected from monophosphate, diphosphate, triphosphate, OP(W)(OH)₂, —OP(W)(OH)OP(W)(OH)₂, —OP(W)(OH)OP(W)(OH)OP(W)(OH)₂, —OS(O)₂aryl(H),

—OS(O)₂aryl(CH₃), —P(W)(OH)₂, —OP(W)(OH)OS(O)₂(OH), —OP(W)(OH)Z, —P(W)(OH)OP(W)(OH)₂, —O(CH₂)₁₋₄OP(W)(OH)₂, —OS(W)(OH)₂, —OP(W)(OH) CH₂OP(W)(OH)₂, —OP(W)(OH)OP(W)O(CH₂)₁₋₄R₁₈, —(CH₂)₀₋₄COOH, —(CH₂)₀₋₄S(O)(OH)₂, —(CH₂)₀₋₄C(O)NHOH and —(CH₂)₀₋₄B(OH)₂;

wherein X is selected from O, S, N(R₃) and CH₂;

wherein W is selected from O and S;

wherein R₃ is selected from H and CH₃;

wherein R₄, R₅, R₁₄, R₁₅, R₁₈ are independently selected from H, OH, SH, CH₂, CH₃, OR₆, SR₆, CH₂CH₃, lower alkyl branched and unbranched (C₁-C₆), XC(O)lower alkyl, —XC(O)CH₂Ph, —P(W)(OH)₂, —XC(O)PhR₁₁, —OP(O)(OH)O, OCH₃, N(R₁₁, R₁₇), —O(C)n R₁₁, R₁₇O—, N(H or independently lower alkyl)₂₋₃ and COOR₁₁, —OC(W)NH(CH₂)₁₋₆NH₂, —OC(W)NH(CH₂)₁₋₆R₄₄

wherein R₄₄ is H, OH, SH, CH₂, CH₃, OR₆, SR₆, CH₂CH₃, lower alkyl branched and unbranched (C₁-C₆), XC(O)lower alkyl, —XC(O)CH₂Ph, —P(W)(OH)₂, —XC(O)PhR₁₁, —OP(O)(OH)O, OCH₃, N(R₁₁, R₁₇), —O(C)n R₁₁, R₁₇O—, N(H or independently lower alkyl)₂₋₃ or COOR₁₁, —OC(W)NH(CH₂)₁₋₆NH₂;

wherein Z is selected from piperidine, morpholine, piperazine, N-methyl-piperazine, N(R₁₆)(R₁₇); and

wherein R₆, R₁₁, R₁₆ and R₁₇ are selected independently from H, OH, SH, CH₂, CH₃, OCH₃, COOR, N(R₁₂)(R₁₃), CH₂CH₃, lower alkyl branched and unbranched (C₁-C₆), XC(O)lower alkyl, —XC(O)CH₂Ph, —P(W)(OH)₂, —XC(O)PhR₁₂, —OP(O)(OH)O, OCH₃, N(R₁₂, R₁₃), —O(C)n R₁₂, R₁₃O—, N(H or independently lower alkyl)₂₋₃ and COOR₁₂; and

wherein R₁₂ and R₁₃ are selected independently from H, OH, SH, CH₂, CH₃, OCH₃ and CH₂CH₃.

In certain embodiments, R₁ in Formula Nt-1 is selected from

In certain embodiments, R₁ in Formula Nt-1 is selected from the compounds listed in Table 1.

Table 1. R₁ group of nucleotide derivatives of Formula Nt-1

TABLE 1 R₁ group of nucleotide derivatives of Formula Nt-1 R₁

wherein Q₁, Q₂, Q₃ and Q₄ are independently selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₆NH₂, —(CH₂)₁₋₄aryl, S(O)₁₋₂ Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c- C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein Y is selected from C or N.

wherein Q₂, Q₃ and Q₄ are independently selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein Y or W are independently selected from O, S, C or N.

wherein Q₃₀ and Q₄ are independently selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein Y or W are independently selected from O, S, or N.

wherein Q₄ is selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein Y or W are independently selected from O, S, or N.

wherein Q₇ is selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein W is selected from O, S, C or N.

wherein Q₇ and Q₈ are independently selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein W is selected from O, S, C or N.

wherein Q₂, Q₃, Q₇ and Q₈ are independently selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein W is selected from O, S, C or N.

wherein Q₇ is selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein W is selected from O, S, C or N.

wherein Q₂, Q₃ and Q₇ are independently selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; and wherein W is selected from O, S, C or N.

wherein Q₂, Q₃ and Q₄ are independently selected from H, CH₃, —SCH₃, N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, unsubstituted or substituted branched or unbranched lower alkyl (C₁-C₁₀), substituted or unsubstituted aryl, —(CH₂)₁₋₄aryl, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂; wherein Q₆ is selected from Me and Ph; wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c- C₄H₇, c-C₄H₇, c-C₅H₉, c-C₆H₁₀ and —CH₂CH₂CH₂CH₂—; wherein W is selected from O and S; wherein Y is selected from C and N, and when Y is nitrogen, R₅₀ is not present, and when Y is carbon, R₅₀ is selected from H, CH₃, —SCH₃, —N(R₁₉)(R₂₀), F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), aryl, —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₄H₇, COOH, c-C₄H₇, c-C₅H₉, c- C₆H₁₀ and —CH₂CH₂CH₂CH₂—; wherein Q₆ is selected from Me and Ph; and wherein R₁₉ and R₂₀ are independently selected from H, CH₃, —SCH₃, F, Cl, Br, I, —OCH₃, O, S, branched or unbranched lower alkyl (C₁-C₁₀), —(CH₂)₁₋₄aryl, SCH₃, S(O)₁₋₂Q₆, COOCH₃, COOEt, —(CH₂)₀₋₄Ph, c-C₄H₇, c- C₄H₇, c-C₅H₉, c-C₆H₁₀, —CH₂CH₂CH₂CH₂ and aryl.

wherein W is selected from O or S; wherein Q₃ is selected from substituted or unsubstituted lower alkyl branched and unbranched (C₁-C₁₅), substituted or unsubstituted aryl, substituted or unsubstituted furan, substituted or unsubstituted thiophene, substituted or unsubstituted phenyl, substituted or unsubstituted pyridine and substituted or unsubstituted naphthalene; and wherein Q₄ is selected from O, S, CH₃, SCH₃, H, Br, F, Cl and I.

wherein W is selected from O or S; wherein R is derived from the side-chains of the 21 naturally occurring amino acids; wherein Q is selected from substituted or unsubstituted lower alkyl branched and unbranched (C₁-C₁₅), substituted or unsubstituted aryl, substituted or unsubstituted furan, substituted or unsubstituted thiophene, substituted or unsubstituted phenyl, substituted or unsubstituted pyridine and substituted or unsubstituted naphthalene; and wherein Q₄ is selected from O, S, CH₃, SCH₃, H, Br, F, Cl and I.

wherein W is selected from O or S; wherein Q₃₀ is selected from substituted or unsubstituted lower alkyl branched and unbranched (C₁-C₁₅), substituted or unsubstituted aryl, substituted or unsubstituted furan, substituted or unsubstituted thiophene, substituted or unsubstituted phenyl, substituted or unsubstituted pyridine and substituted or unsubstituted naphthalene; and wherein Q₄ is selected from O, S, CH₃, SCH₃, H, Br, F, Cl and I.

The substituents in the substituted groups described herein, for example, “substituted alkyl”, “substituted aryl”, “substituted furan”, “substituted thiophene”, “substituted alkyl”, “substituted phenyl”, “substituted pyrimidine” or “substituted naphthalene” may be the same or different with one or more selected from the groups described in the present application and hydrogen, halogen, methyl, amido, acetyl, nitro (—NO₂), hydroxyl (—OH), oxo (═O), thio (═S), OCH₃, methylene dioxy, CN, NO₂, COOH, SO₃H, S(O)₁₋₂CH₃, S(O)₁₋₂aryl, SCH₃, OH, N(R)₁₋₂, COOCH₃, OC(O)CH₃, SH, sulfonyl, sulfonamido, sulfate, cyano, azido, trifluoromethyl (—CF₃), methoxy (—OCH₃), tert-butyl carbamate (-Boc) or optionally substituted groups selected from alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy, arylalkyl, ethereal, carboxy, hydroxyl, heteroaryl, heteroarylalkyl, sulfonyl, and heterocyclic. A “substituted” functionality may have one or more than one substituent.

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-2 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-3 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-4 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-5 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-6 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-7 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-8 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-9 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-10 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-11 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-12 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-13 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-14 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-15 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-16 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-17 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-18 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-19 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-20 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-21 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-22 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-23 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-24 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-25 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-26 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-27 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-28 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-29 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-30 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-31 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-32 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-33 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-34 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-35 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-36 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-37 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-38 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-39 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-40 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-41 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-42 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-43 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-44 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-45 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-46 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-47 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-48 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-49 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-50 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-51 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-52 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-53 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-54 having the following structure:

In certain embodiments, the nucleotide derivative comprises a compound of Formula Nt-55 having the following structure:

In certain embodiments, the nucleotide derivative can be a salt, stereoisomer or a comestible form of compounds of Formulas Nt-1 to Nt-55 and/or the compounds listed in Tables 2 and 6-14.

TABLE 2 Nucleotide derivatives Chemical Name Adenosine 3′,5′-diphosphate (ADP) 2′-/3′-O-(N′-Methyl-anthraniloyl) adenosine-5′-O-monophosphate (MANT-AMP) 2′-Deoxy-3′-O-(N′-methylanthraniloyl)adenosine-5′-O-monophosphate Adenosine-5′-O-monophosphorothioate (5′-AMPS) 1,N6-Ethenoadenosine-5′-O-monophosphate (ε-5′-AMP) 2′-Deoxy-N6-methyl adenosine 3′,5′-diphosphate 2′-Deoxyadenosine-3′,5′-O-bisphosphate Adenosine 5′-phosphosulfate N6-Cyclopentyladenosine-5′-O-monophosphate (6-cPe-5′-AMP) Adenosine 5′-O-thiomonophosphate 2′-Deoxyadenosine-5′-O-monophosphorothioate (5′-dAMPS) 2′-/3′-O-(2-Aminoethyl carbamoyl)adenosine-5′-O-monophosphate (2′-/3′- AEC-5′-AMP) N6-Methyladenosine 5′-monophosphate 2′-Deoxyadenosine 5′-monophosphate 2′-Deoxy-1,N6-ethenoadenosine-5′-O-monophosphate 7-Deazaadenosine-5′-O-monophosphate (7-CH-5′-AMP/5′-TuMP) 8-Chloroadenosine-5′-O-monophosphate (8-Cl-5′-AMPS) N6-Benzoyladenosine-5′-O-monophosphate (6-Bnz-5′-AMP) 6-Thioguanosine-5′-O-monophosphate (6-T-5′-GMP) 2′-Deoxyguanosine-5′-O-monophosphorothioate 2′-,3′-O-(N′-Methylanthraniloyl)guanosine-5′-O-monophosphate (MANT-5′-GMP) (Fluorescent analogue of 5′-GMP) Guanosine-5′-monophosphorothioate (5′-GMPS) 2′-Deoxy-3′-O-(N′-methylanthraniloyl) guanosine-5′-O-monophosphate Guanosine-5′-O-(2-thiodiphosphate) 2′-Deoxyguanosine-3′,5′-O-bisphosphate 2′-Deoxyguanosine-5′-O-monophosphorothioate (5′-dGMPS) N-Lactoyl-5′-GMP N-Acetyl-5′-GMP Guanosine 5′-diphosphate 2′-/3′-O-(2-Aminoethylcarbamoyl) guanosine-5′-O-monophosphate (2′-/ 3′-AEC-5′-GMP) Guanosine 5′-triphosphate 2′-Deoxyguanosine 5′-monophosphate Guanosine 5′-monophosphomorpholidate 4-morpholine-N,N′- dicyclohexylcarboxamidine 8-Bromoguanosine-5′-O-monophosphate (8-Br-5′-GMP) 2-Amino-6-chloropurineriboside-5′-O-monophosphate (2-NH2-6-Cl-5′-PuMP) 6-Chloropurineriboside-5′-O-monophosphate 6-Thioinosine Phosphate Inosine Triphosphate (ITP) 2′-Deoxyinosine-5′-O-monophosphorothioate Inosine 5′-diphosphate (IDP) Uridine 5′-monophosphomorpholidate 4-morpholine-N,N′- dicyclohexylcarboxamidine 2′-Deoxyuridine-5′-O-monophosphosphorothioate (5′-dUMPS) Ribavirin 5′-Monophosphate

In certain embodiments, the nucleotide derivatives of the present disclosure comprise a salt of the nucleotide derivative, for example, but not limited to, an acetate salt or a formate salt. In certain embodiments, the nucleotide derivative salt comprises an anion (−) (for example, but not limited to, Cl⁻, O²⁻, CO₃ ²⁻, HCO₃ ⁻, OH⁻, NO₃ ⁻, PO₄ ³⁻, SO₄ ²⁻, CH₃COO—, HCOO⁻ and C₂O₄ ²⁻) bonded via an ionic bond with a cation (+) (for example, but not limited to, Al³⁺, Ca²⁺, Na⁺, K⁺, Cu²⁺, H⁺, Fe³⁺, Mg², NH₄ ⁺ and H₃O⁺). In other embodiments, the nucleotide derivative salt comprises a cation (+) bonded via an ionic bond with an anion (−).

In certain embodiments, the nucleotide derivatives of the present application are identified through in silico modeling of the feline T1R1/T1R3 receptor (“Umami receptor”), wherein the nucleotide derivatives of the present application comprise a structure that fits within a binding site of the feline T1R1/T1R3 receptor.

In certain embodiments, the nucleotide derivatives of the present application are identified through an in vitro assay, wherein the nucleotide derivatives activate a feline T1R1/T1R3 receptor expressed by cells in vitro. In certain embodiments, the nucleotide derivatives activate the receptor alone, or in combination with other T1R1/T1R3 binding agents, such as the nucleotides, amino acids and transmembrane compounds described herein, and/or in combination with amino acids described herein that bind to one or more other receptors. In certain embodiments the in vitro assay comprises the in vitro assays described in the Examples section of the present application.

2.1 T1R1 Nucleotide Binding Site

The present application also provides for compositions that modulate an umami receptor, for example, a T1R1/T1R3 receptor, wherein the compositions interact with one or more amino acids in a Venus Flytrap domain of the umami receptor. In certain embodiments, the Venus Flytrap domain (VFT) is present in T1R1. In certain embodiments, the amino acids that the composition interacts with comprise one or more of Thr449, Ser172, Glu170, Glu301, His71, His47, Arg277, His308, Asn69, Asn302, Ser306, Ser384, Asp302, Ser306, and Ala380.

In one non-limiting embodiment, the composition comprises a nucleotide and/or nucleotide derivative, wherein the nucleotide and/or nucleotide derivative interacts with one, two, three, four, five, six, seven, eight or more of His71, Arg277, His308, Ser306, Ser384, Ala380, His47, Asn69, and Asp302 of T1R1.

In another non-limiting embodiment, the composition comprises a nucleotide and/or nucleotide derivative, wherein the nucleotide and/or nucleotide derivative interacts with one, two, three, four or more of Met383, Ser385, Ile309, Ser107, and Asp49 of T1R1.

When binding a nucleotide and/or nucleotide derivative, the amino acid residues of the VFT may coordinate the phosphates, diphosphates, triphosphates, bisphosphates, phosphate mimetics (e.g. acidic functionalities such as COOH, SO₃H, NHOH, etc.), of a nucleotide and/or nucleotide derivative, and include one or more of His71, His47, Arg277, His308, Ile309, Asn69, Ser107, and Asp49 of T1R1.

In certain embodiments, at least one phosphate of the nucleotide and/or nucleotide derivative interacts with one, two, three, four or more of His71, His47, Arg277, His308, and Asn69 of T1R1. In a non-limiting example, binding of a nucleotide and/or nucleotide derivative to the VFT involves interactions between negatively charged groups of amino acids in the phosphate binding region of VFT and a nucleotide and/or nucleotide derivative phosphate.

The amino acid residues of the VFT may coordinate the sugar atoms of a nucleotide and/or nucleotide derivative (or, for example, a modified sugar or sugar replacement), and can include Asp302 and/or Ser306 of T1R1.

In certain embodiments, at least one sugar molecule of the nucleotide and/or nucleotide derivative interacts with amino acids Asp302 and/or Ser306 of T1R1.

The amino acid residues of the VFT may coordinate a nitrogenous base of a nucleotide and/or nucleotide derivative, and can include one or more of Ser384, Ser385, Ala380, Met383, Glu170 and Asp302 of T1R1.

In certain embodiments, the nitrogenous base of the nucleotide and/or nucleotide derivative interacts with one, two or more of Ser384, His308, and Ala380 of T1R1.

In other non-limiting embodiments, a sugar molecule of the nucleotide and/or nucleotide derivative interacts with Asp302 of the VFT, wherein Asp302 is oriented to simultaneously coordinate the zwitterionic backbone nitrogen of a bound amino acid and the sugar of the nucleotide or nucleotide derivative.

In certain embodiments, the composition interacts with the umami Venus Flytrap domain (VFT) according to any combination of interactions described herein, for example, one, two, three or more of the interactions. The interactions between a nucleotide and/or nucleotide derivative and the VFT may further include additional hydrophobic interactions that add to the interaction energy of the nucleotide and/or nucleotide derivative to the VFT.

In certain embodiments, the interaction between the composition and the one or more amino acids comprises one or more hydrogen bond, covalent bond, non-covalent bond, salt bridge, physical interaction, and combinations thereof. The interactions can also be any interaction characteristic of a ligand receptor interaction known in the art. Such interactions can be determined by, for example, site directed mutagenesis, x-ray crystallography, x-ray or other spectroscopic methods, Nuclear Magnetic Resonance (NMR), cross-linking assessment, mass spectroscopy or electrophoresis, displacement assays based on known agonists, structural determination and combinations thereof. In certain embodiments, the interactions are determined in silico, for example, by theoretical means such as docking a compound into the VFT domain using molecular docking, molecular modeling, molecular simulation, or other means known to persons of ordinary skill in the art.

The present application also provides for methods of identifying a compound that modulates the activity of an umami receptor, for example, a T1R1, wherein the compound is identified based on its ability to interact with one or more of the amino acids described herein that are present in the VFT domain of T1R1.

In certain embodiments the method comprises contacting a test agent with a feline T1R1 umami receptor, detecting an interaction between the test agent and one more amino acids in a VFT interacting site of the feline T1R1 umami receptor, and selecting as the compound, a test agent that interacts with one or more of the amino acids.

3. TRANSMEMBRANE COMPOUNDS

The present disclosure relates to flavor compositions that include at least one transmembrane compounds. In certain embodiments, the transmembrane compound is an umami taste enhancing compound. The transmembrane compounds disclosed herein were identified through the in silico modeling of putative agonists within the transmembrane region of T1R1 of the feline T1R1/T1R3 receptor (“Umami receptor”). Accordingly, in certain embodiments, a transmembrane compound is a composition that interacts with (e.g., binds to) a region of a T1R1 comprising the transmembrane domain of T1R1. In certain embodiments, such interactions with the transmembrane domain of T1R1 agonizes the T1R1/T1R3 or umami receptor. In other embodiments, the transmembrane compound acts synergistically with other T1R1 agonists or modulators to modulate the activity of the T1R1/T1R3 or umami receptor.

The flavor compositions can be used to enhance or modify the palatability, taste or flavor of pet food product. The flavor compositions can include combinations of compounds, for example, combinations of transmembrane compounds, and/or nucleotides, and/or nucleotide derivatives and/or amino acids, and can be added to pet food product compositions in various delivery system formats.

3.1 Amine Derivatives I

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-1 having the following structure:

wherein X₁ is selected from the group consisting of O and S;

wherein n₁ is 1-3;

wherein n₂ is 0-4 (wherein when n is 0, a chemical bond is present);

wherein n₄ is 0-3;

wherein R₁, R₂, and R₃ are independently selected from the group consisting of H, ═O, ═S, branched or unbranched and substituted or unsubstituted lower alkyl (C₁-C₈), and R₅;

wherein R₄ is selected from the group consisting of H, branched or unbranched lower alyl (C₁-C₈), and (CH₂)_(n2)aryl;

wherein R₅ is selected from the group consisting of H, CH₃, CH(CH₃)₂, CH₂CH(CH₃)₂, CH(CH₃)CH₂CH₃, CH₂CH₂SCH₃, CH₂SH, CH₂SeH, CH₂OH, CH(OH)CH₃, CH₂C(O)NH₂, CH₂CH₂C(O)NH₂, CH₂CH₂CH₂NHC(NH)NH₂, CH₂(1H-imidazole-yl), CH₂(CH₂)₂CH₂NH₂, CH₂COOH, CH₂CH₂COOH, CH₂C₆H₅, CH₂(4-hydroxphenyl), CH₂[3-yl-(1H-indol)], CH₂(cyclo-pentyl), CH₂(cyclo-hexyl), CH₂(indanyl), independently branched or unbranched lower alkyl (C₁-C₁₀), (CH₂)₀₋₄PH, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀, phenyl, biaryl, (CH₂)_(n2)aryl, pyridine, thiophene, CH₂Ph, CH₂pyridine, and CH₂thiophene,

-   -   the aryl and alkyl (both branched and unbranched) groups may         optionally be substituted by methyl, OH, SH, OCH₃, SCH₃, COOH,         COOR₁₃, S(O)n₄R₁, C(O)R₁₁, C(O)NR₁₁R₁₂, CN, NR₁₁R₁₂,         NR₁₁C(O)R₁₂, aryl, methylenedioxy, alkyl (C₁-C₅),         CH₂SSCH₂CH(COOH)(NH₂), Halogen (F, Cl, Br, I), NO₂, NHC(═NH)NH₂,         CHO, CF₃, P(═X₁)(OR₁)₂, OP(═X₁)(OR₁)₂;

wherein R₅ and R₆ may be joined to form cyclic rings such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, (i.e. spiro rings); wherein R₆ is selected from the group consisting of H, and branched or unbranched lower alkyl (C₁-C₄);

wherein R₇ is selected from the group consisting of H, AA, OH, O, branched or unbranched lower alkyl (C₁-C₆), O(CH₂)_(n1)aryl, NR₁₁R₁₂, N(R₁₄)OH, C(R₈)(R₉), aryl, and heteroaryl;

wherein R₈ and R₉ are independently selected from the group consisting of H, branched or unbranched lower alkyl(C₁-C₆), aryl, alkyl-aryl, and alkyl-heteroaryl;

wherein R₁₁ and R₁₂ are independently selected from the group consisting of H, CH₃, branched or unbranched lower alkyl (C₁-C₆), phenyl, aryl, and (CH₂)_(n1)aryl;

wherein R₁₃ is selected from the group consisting of H, CH₃, CH₂CH₃, CH₂aryl, and tert-butyl;

wherein R₁₄ is selected from the group consisting of H and CH₃; and

wherein AA is selected from the group consisting of a naturally occurring alpha amino acid or (R) or (S)-configuration (i.e., proteogenic amino acids).

In one non-limiting embodiment, all asymmetric configurations are contemplated for the presently disclosed formulations.

In certain embodiments, aryl has its standard chemical meaning and may comprise the following, but is not limited to, Ph, pyridine, thiophene, furan, napthyl, indole, benzothiophene, benzofuran, quinolone, isoquinoline, pyrrole, N-(methyl)pyrolle, imidazole, thiazole, pyrimidine, isoxazole, oxazole, isoindole, indolizine, purine, pyrazine, and pyridazine.

In certain embodiments, attachments of the aryl group may be placed at various carbon centers, as would be understood by one of ordinary skill in the art.

In other non-limiting embodiments, when the compound comprises two aryl rings (e.g., phenyls), either one or both rings may be substituted by a biaryl ring system. Such biaryl ring systems include, for example, phenyl-phenyl, phenyl-pyridyl, phenyl-thiophene, thiophene-thiophene, and phenyl-furan.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-2 having the following structure:

wherein R₄, R₅, R₆, R₇, X₁, and n₁ are as defined for Formula Tm-1.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-20 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-21 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-22 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-23 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-24 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-25 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-26 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-27 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-28 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-29 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-30 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-31 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-32 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-33 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-34 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-35 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-36 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-37 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-38 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-39 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-40 having the following structure:

3.2 Amine Derivatives II

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-3 having the following structure:

wherein X₁ is selected from the group consisting of O and S;

wherein n₁ and n₂ are 0-4 (wherein when n₁ and/or n₂ is 0, a chemical bond is present);

wherein R₁ is selected from the group consisting of branched or unbranched lower alkyl (C₁-C₁₀), (CH₂)_(n2)Ph, c-C₃H₅, c-C₄H₇, c-C₅H₉, c-C₆H₁₀, Phenyl, (CH₂)_(n2)aryl, Ph, pyridine, thiophene, CH₂Ph, CH₂pyridine, CH₂thiophene, O-aryl, Ph, pyridine, thiophene, furan, napthyl, indole, benzothiophene, benzofuran, quinolone, isoquinoline, pyrrole, N-(methyl)pyrolle, imidazole, thiazole, pyrimdine, isoxazole, oxazole, isoindole, indolizine, purine, pyrazine, pyridazine, O-alkyl(C₁-C₆), biaryl, and OR₁ (e.g., carbamates),

-   -   the aryl and alkyl (both branched and unbranched) groups may         optionally be substituted by methyl, OH, SH, OCH₃, SCH₃, COOH,         COOR₁₃, S(O)n₄R₁, C(O)R₁₁, C(O)NR₁₁R₁₂, CN, NR₁₁R₁₂,         NR₁₁C(O)R₁₂, aryl, methylenedioxy, alkyl (C₁-C₅),         CH₂SSCH₂CH(COOH)(NH₂), Halogen (F, Cl, Br, I), NO₂, NHC(═NH)NH₂,         CHO, CF₃, P(═X₁)(OR₁)₂, or OP(═X₁)(OR₁)₂;

wherein R₂ is selected from the group consisting of H, CH₃, (CH₂), and aryl;

wherein R₃, R₄, and R₅ are independently selected from the group consisting of H, branched or unbranched lower alkyl (C₁-C₈), (CH₂)_(n2)aryl, and R₁;

wherein R₁₁ and R₁₂ are independently selected from the group consisting of H, CH₃, branched or unbranched lower alkyl (C₁-C₆), phenyl, aryl, and (CH₂)_(n1)aryl;

wherein R₁₃ is selected from the group consisting of H, CH₃, CH₂CH₃, CH₂aryl, and tert-butyl;

In certain embodiments, aryl has its standard chemical meaning and may comprise, but is not limited to, Ph, pyridine, thiophene, furan, napthyl, indole, benzothiophene, benzofuran, quinolone, isoquinoline, pyrrole, N-(methyl)pyrolle, imidazole, thiazole, pyrimidine, isoxazole, oxazole, isoindole, indolizine, purine, pyrazine, and pyridazine.

In certain embodiments, attachments of the aryl group may be placed at various carbon centers, as would be understood by one of ordinary skill in the art.

In certain embodiments, asymmetric centers may be of either (R) or (S) configuration, as would be understood by one of ordinary skill in the art.

In one non-limiting embodiment, all asymmetric configurations are contemplated for the presently disclosed formulations.

In other non-limiting embodiments, when the compound comprises two aryl rings (e.g., phenyls), either one or both rings may be substituted by a biaryl ring system. Such biaryl ring systems include, for example, phenyl-phenyl, phenyl-pyridyl, phenyl-thiophene, thiophene-thiophene, and penyl-furan.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-4 having the following structure:

wherein R1 and R2 are independently selected from the group consisting of aryl, cyclo-alkyl (e.g., cyclo-propyl, cyclo-butyl, cyclo-pentyl, cyclo-hexyl, cyclo-heptyl), and hetero aryl, (for example, but not limited to, Ph, pyridine, thiophene, furan, napthyl, indole, benzothiophene, benzofuran, quinolone, isoquinoline, pyrrole, N-(methyl)pyrolle, imidazole, thiazole, pyrimidine, isoxazole, oxazole, isoindole, indolizine, purine, pyrazine, and pyridazine);

wherein the aryl groups or cyclo-alkyl groups may optionally be substituted by methyl, OH, SH, OCH₃, SCH₃, COOH, COOR₁₃, S(O)n₄R₁, C(O)R₁₁, C(O)NR₁₁, R₁₂, CN, NR₁₁C(O)R₁₂, aryl, methylenedioxy, alkyl (C₁-C₅), CH₂SSCH₂CH(COOH)(NH₂), Halogen (F, Cl, Br, I), NO₂, NHC(═NH)NH₂, CHO, CF₃, P(═X₁)(OR₁)₂ or OP(═X₁)(OR₁)₂;

wherein the cyclo-alkyl groups may optionally contain hetero atoms within the rings (e.g., O, N and/or S), for example, piperidine, piperazine, tetrahydrothiophene, pyran, pyrrolidine, or tetrahydrofuran;

wherein n₁=0-4;

wherein R₁₁, R₁₂, R₁₃, and X₁ are as defined herein; and

wherein n₄=0-4.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-41 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-42 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-43 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-44 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-45 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-46 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-47 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-48 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-49 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-50 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-51 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-52 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-53 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-54 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-55 having the following structure:

3.3 Parabanic Acid Derivatives

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-6 having the following structure:

wherein R¹ or R² are selected independently from the group consisting of a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, hydroxyl, hydrogen, substituted or unsubstituted ethereal, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thienyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted quinolyl, substituted or unsubstituted indenyl, or substituted or unsubstituted indanyl.

In certain embodiments, R¹ or R² can comprise the following structure:

wherein R³, R⁴, R⁵, R⁶ and R⁷ are selected independently from the group consisting of hydrogen, halogen, cyano, azido, hydroxyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonamido, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted amido, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted ethereal, substituted or unsubstituted carboxy, substituted or unsubstituted acyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thienyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted quinolyl, or substituted or unsubstituted heteroarenyl, or substituted or unsubstituted indene, or substituted or unsubstituted indanyl. In certain embodiments, two or more of R³, R⁴, R⁵, R⁶ and R⁷ may be taken together to form a ring, which can be heterocyclic (i.e., contain one or more heteroatoms) or which can be entirely carbocyclic and which can independently be saturated or unsaturated.

The substituents in the substituted groups described herein, for example, “substituted ethereal”, “substituted carboxy”, “substituted acyl”, “substituted sulfonyl”, “substituted alkyl”, “substituted alkenyl”, “substituted cycloalkyl”, “substituted cycloalkalkyl”, “substituted arylalkyl”, “substituted aryl”, “substituted heterocyclic”, “substituted heteroarylalkyl,” “substituted heteroaryl”, “substituted naphthyl”, “substituted phenyl”, “substituted thienyl”, “substituted benzothienyl”, “substituted pyridyl”, “substituted indolyl”, “substituted isoquinolyl”, “substituted quinolyl”, “substituted benzothiazolyl”, “substituted heteroaryl,” “substituted indenyl” or “substituted indanyl” may be the same or different with one or more selected from the groups described in the present application and hydrogen, halogen, amido, acetyl, nitro (—NO₂), hydroxyl (—OH), oxo (═O), thio (═S), sulfonyl, sulfonamido, sulfate, thio, cyano, azido, trifluoromethyl (—CF₃), methoxy (—OCH₃), tert-butyl carbamate (-Boc) or optionally substituted groups selected from alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy, arylalkyl, ethereal, carboxy, hydroxyl, heteroaryl, sulfonyl, and heterocyclic. A “substituted” functionality may have one or more than one substituent.

In one non-limiting embodiment, R¹ and R² are unsubstituted phenyl groups.

In one non-limiting embodiment, R¹ is an unsubstituted phenyl group and R² is a substituted phenyl group.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-7 having the following structure:

wherein R₁ and R₂ are as defined above for Formula Tm-6.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-5 having the following structure:

wherein X₁ is selected from the group consisting of O, N(R₃), S;

wherein X₂ and X₃ are selected independently from the group consisting of O and S;

wherein R₃ is selected from the group consisting of H and branched or unbranched lower alkyl (C₁-C₄); and

wherein R₁ and R₂ are as defined above for Formula Tm-6, and may also be (CH₂)_(n)C(═X₁)R₁, (CH₂)_(n)C(═X₁)R₂, wherein X₁ is O or S, and n is 0-4.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-9 having the following structure:

wherein X₁₋₅ are selected independently from the group consisting of O and S; and wherein R₁ and R₂ are as defined above for Formula Tm-6.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-10 having the following structure:

wherein X₁₋₅ are selected independently from the group consisting of O and S; and wherein R₁ and R₂ are as defined above for Formula Tm-6.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-11 having the following structure:

wherein X₁₋₃ are selected independently from the group consisting of O and S; and wherein R₁ and R₂ are as defined above for Formula Tm-6.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-56 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-57 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-58 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-59 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-60 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-61 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-62 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-63 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-64 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-65 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-66 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-67 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-68 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-69 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-70 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-71 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-72 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-73 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-74 having the following structure:

3.4 Imidazopyridinone Derivatives

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-19 having the following structure:

wherein R¹ or R² are selected independently from the group consisting of a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, hydroxyl, hydrogen, substituted or unsubstituted ethereal, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thienyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted quinolyl, substituted or unsubstituted indenyl, or substituted or unsubstituted indanyl.

In certain embodiments, R¹ or R² can comprise the following structure:

wherein R³, R⁴, R⁵, R⁶ and R⁷ are selected independently from the group consisting of hydrogen, halogen, cyano, azido, hydroxyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonamido, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted amido, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted ethereal, substituted or unsubstituted carboxy, substituted or unsubstituted acyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thienyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted quinolyl, or substituted or unsubstituted heteroarenyl, or substituted or unsubstituted indene, or substituted or unsubstituted indanyl. In certain embodiments, two or more of R³, R⁴, R⁵, R⁶ and R⁷ may be taken together to form a ring, which can be heterocyclic (i.e., contain one or more heteroatoms) or which can be entirely carbocyclic and which can independently be saturated or unsaturated.

The substituents in the substituted groups described herein, for example, “substituted ethereal”, “substituted carboxy”, “substituted acyl”, “substituted sulfonyl”, “substituted alkyl”, “substituted alkenyl”, “substituted cycloalkyl”, “substituted cycloalkalkyl”, “substituted arylalkyl”, “substituted aryl”, “substituted heterocyclic”, “substituted heteroarylalkyl,” “substituted heteroaryl”, “substituted naphthyl”, “substituted phenyl”, “substituted thienyl”, “substituted benzothienyl”, “substituted pyridyl”, “substituted indolyl”, “substituted isoquinolyl”, “substituted quinolyl”, “substituted benzothiazolyl”, “substituted heteroaryl,” “substituted indenyl” or “substituted indanyl” may be the same or different with one or more selected from the groups described in the present application and hydrogen, halogen, amido, acetyl, nitro (—NO₂), hydroxyl (—OH), oxo (═O), thio (═S), sulfonyl, sulfonamido, sulfate, thio, cyano, azido, trifluoromethyl (—CF₃), methoxy (—OCH₃), tert-butyl carbamate (-Boc) or optionally substituted groups selected from alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, aryl, aryloxy, arylalkyl, ethereal, carboxy, hydroxyl, heteroaryl, sulfonyl, and heterocyclic. A “substituted” functionality may have one or more than one substituent.

In one non-limiting embodiment, R¹ and R² are unsubstituted phenyl groups.

In one non-limiting embodiment, R¹ is an unsubstituted phenyl group and R² is a substituted phenyl group.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-12 having the following structure:

wherein Y is selected from the group consisting of O, S and N(R₄);

wherein X₁₋₃ are selected independently from the group consisting of C, O, N and S;

wherein R₃ is a substituted or unsubstituted aromatic substituent, wherein the substituent is, for example, H, OR₄, S(O)_(n)R₄, N(R₄)(R₅), CN, COOH, COOR, C(O)N(R₄)(R₅), SO₂N(R₄)(R₅), halogen (e.g., Cl, Br, F, I), branched or unbranched substituted or unsubstituted lower alkyl (C1-C8), aryl, biaryl, P(O)(OH)₂, NHOH, B(OH)₂, C(═NH)NH2, NHC(═NH)NH₂, NO₂, CF3, —OCH₂O— (i.e., methylenedioxy), branched or unbranched substituted or unsubstituted lower alkyne (C2-C6), branched or unbranched substituted or unsubstituted lower alkene (C2-C6), (CH₂)_(n)aryl;

wherein R4 and R5 are independently H, branched or unbranched substituted or unsubstituted lower alkyl (C1-C8), branched or unbranched substituted or unsubstituted lower alkyne (C2-C6), branched or unbranched substituted or unsubstituted lower alkene (C2-C6), aryl, (CH2)_(n)aryl;

wherein n is 0-4; and

wherein R₁ and R₂ are as described for Formula Tm-19.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-13 having the following structure:

wherein Y is selected from the group consisting of O, S and N(R₄);

wherein X₁₋₄ are selected independently from the group consisting of C, O, N and S;

wherein R₃ is a substituted or unsubstituted aromatic substituent, wherein the substituent is, for example, H, OR₄, S(O)_(n)R₄, N(R₄)(R₅), CN, COOH, COOR, C(O)N(R₄)(R₅), SO₂N(R₄)(R₅), halogen (e.g., Cl, Br, F, I), H, branched or unbranched substituted or unsubstituted lower alkyl (C1-C8), aryl, biaryl, P(O)(OH)₂, NHOH, B(OH)₂, C(═NH)NH₂, NHC(═NH)NH₂, NO₂, CF3, —OCH₂O— (i.e., methylenedioxy), branched or unbranched substituted or unsubstituted lower alkyne (C2-C6), branched or unbranched substituted or unsubstituted lower alkene (C2-C6), (CH2)_(n)aryl;

wherein R4 and R5 are independently H, branched or unbranched substituted or unsubstituted lower alkyl (C1-C8), branched or unbranched substituted or unsubstituted lower alkyne (C2-C6), branched or unbranched substituted or unsubstituted lower alkene (C2-C6), aryl, (CH2)_(n)aryl;

wherein n is 0-4; and

wherein R₁ and R₂ are as described for Formula Tm-19.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-14 having the following structure:

wherein X is selected from the group consisting of C, O, N and S; and wherein R₁₋₃ are as described for Formulas Tm-19, Tm-12 and Tm-13.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-15 having the following structure:

wherein R₁₋₂ are as described for Formulas Tm-19, Tm-12 and Tm-13.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-16 having the following structure:

wherein X is selected from the group consisting of C, O, N and S; and

wherein R₁₋₂ are as described for Formulas Tm-19, Tm-12 and Tm-13.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-17 having the following structure:

wherein R₁₋₂ are as described for Formulas Tm-19, Tm-12 and Tm-13.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-18 having the following structure:

wherein R₂ is as described for Formulas Tm-19, Tm-12 and Tm-13.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-75 having the following structure:

3.5 Pyrimidine-2,4,6-trione Derivatives

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-8 having the following structure:

wherein X₁ is selected from the group consisting of O, N(R₁₂) and S; wherein X₂ and X₃ are selected independently from the group consisting of O and S;

wherein X₄ is selected from the group consisting of NH, N(R₇), and C(R₈, R₉);

wherein n₁ is 0-1, and when n₁ is 0 then a chemical bond is between the two carbons bearing X₂ and X₃;

wherein R₇, R₈, and R₉ are selected independently from the group consisting of H, substituted or unsubstituted branched or unbranched lower alkyl (C₁-C₂₀), aryl, heteroaryl, cyclo-alkyl (C3-C7), and substituted, unsubstituted, branched, or unbranched C(CH2)_(n2)aryl,

-   -   substitution on alkyl and aryl groups include functionality for         those skilled in the art such as OH, NH₂, Halogen, SH, nitro,         aryl, alkene, COOH, COOR, C(O)N(R₄)(R₅), SO₂N(R₄)(R₅), NO2,         P(O)(OH)₂, NHOH, B(OH)₂, C(═NH)NH₂, NHC(═NH)NH₂, NO₂, and CF3;     -   wherein R4 and R5 are independently H, branched or unbranched         substituted or unsubstituted lower alkyl (C1-C8), branched or         unbranched substituted or unsubstituted lower alkyne (C2-C6),         branched or unbranched substituted or unsubstituted lower alkene         (C2-C6), aryl, (CH2)_(n)aryl;

wherein n₂ is 0-10;

wherein R₁₂ is selected from the group consisting of H, branched or unbranched lower alkyl (C₁-C₄); and

wherein R₁ and R₂ are selected independently from the group consisting of a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, hydroxyl, hydrogen, substituted or unsubstituted ethereal, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thienyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted quinolyl, substituted or unsubstituted indenyl, or substituted or unsubstituted indanyl.

In certain embodiments, R₁ and/or R₂ can comprise the following structure:

wherein R³, R⁴, R⁵, R⁶ and R⁷ are selected independently from the group consisting of hydrogen, halogen, cyano, azido, hydroxyl, substituted or unsubstituted sulfonyl, substituted or unsubstituted sulfonamido, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted amido, substituted or unsubstituted heterocyclic, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted ethereal, substituted or unsubstituted carboxy, substituted or unsubstituted acyl, substituted or unsubstituted benzothiazolyl, substituted or unsubstituted pyridyl, substituted or unsubstituted naphthyl, substituted or unsubstituted thienyl, substituted or unsubstituted benzothienyl, substituted or unsubstituted indolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted quinolyl, or substituted or unsubstituted heteroarenyl, or substituted or unsubstituted indene, or substituted or unsubstituted indanyl. In certain embodiments, two or more of R³, R⁴, R⁵, R⁶ and R⁷ may be taken together to form a ring, which can be heterocyclic (i.e., contain one or more heteroatoms) or which can be entirely carbocyclic and which can independently be saturated or unsaturated.

In one non-limiting embodiment, R₁ and R₂ are unsubstituted phenyl groups.

In one non-limiting embodiment, R₁ is an unsubstituted phenyl group and R₂ is a substituted phenyl group.

In one non-limiting embodiment, R₁ and R₂ are as defined above for Formulas Tm-6, Tm-19, Tm-12 and Tm-13, and may also be (CH₂)_(n)C(═X₁)R₁, (CH₂)_(n)C(═X₁)R₂, wherein X₁ is O or S, and n is 0-4.

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-76 having the following structure:

3.6 Additional Transmembrane Compounds

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-77 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-78 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-79 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-80 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-81 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-82 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-83 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-84 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-85 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-86 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-87 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-88 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-89 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-90 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-91 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-92 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-93 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-94 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-95 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-96 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-97 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-98 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-99 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-100 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-101 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-102 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-103 having the following structure:

In certain embodiments, the transmembrane compound comprises a compound of Formula Tm-104 having the following structure:

In certain embodiments of the present disclosure, the transmembrane compound comprises a compound described in Table 3 below.

TABLE 3 Transmembrane compounds. Compound Name 5-bromo-N-(pentan-3-yl)furan-2-carboxamide 3,4-dimethyl-N-(pentan-3-yl)benzamide; N-(1-ethylpropyl)-3,4- dimethylbenzamide 4-methoxy-3-methyl-N-(1-phenylethyl)benzamide N-(2,3-dimethylcyclohexyl) benzo[d][1,3]dioxole-5-carboxamide ethyl 2-(benzo[d][1,3] dioxole-5-carboxamido)-3-methylbutanoate; ethyl N-(1,3-benzodioxol-5 ylcarbonyl) valinate 4-methoxy-3-methyl-N-(pentan-2-yl)benzamide N-(pentan-3-yl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxamide 3,4-dimethoxy-N-(pentan-3-yl)benzamide (E)-methyl 3-methyl-2-(3-(thiophen-2-yl) acrylamido)butanoate (E)-3-(3,4-dimethoxyphenyl)-N-(pentan-2-yl)acrylamide; (2E)-3-(3,4- dimethoxyphenyl)-N-(pentan-2-yl)prop-2-enamide (E)-3-(4-methoxyphenyl)-N-(2-methylcyclohexyl) acrylamide N1-phenethyl-N2-(pyridin-3-ylmethyl)oxalamide; N-(2-phenylethyl)-N′- (pyridin-3-ylmethyl)ethanediamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide 1-(2-bromophenyl)-3-((1R,2S)-2-hydroxy-2,3-dihydro-1H-inden-1-yl)urea N-(benzo[d][1,3]dioxol-5-yl)-2-propylpentanamide 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide (E)-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide 2-((5-(4-(methylthio)phenyl)-2H-tetrazol-2-yl)methyl)pyridine N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide

3.7 Transmembrane Compound Salts

In certain embodiments, the transmembrane compounds of the present disclosure comprise a salt of the transmembrane compound, for example, but not limited to, an acetate salt, a TFA salt or a formate salt. In certain embodiments, the transmembrane compound salt comprises an anion (−) (for example, but not limited to, Cl⁻, F⁻, Br⁻, O²⁻, CO₃ ²⁻, HCO₃ ⁻, OH⁻, NO₃ ⁻, PO₄ ³⁻, SO₄ ²⁻, CH₃COO—, HCOO⁻, C₂O₄ ² and CN⁻) bonded via an ionic bond with a cation (+) (for example, but not limited to, Al³⁺, Ca²⁺, Na⁺, K⁺, Cu²⁺, H⁺, Fe³⁺, Mg²⁺, Ag⁺, NH₄ ⁺, H₃O⁺, Hg₂ ²⁺). In other embodiments, the transmembrane compound salt comprises a cation (+) bonded via an ionic bond with an anion (−).

In certain embodiments, the transmembrane compound can be a salt, stereoisomer or a comestible form of a transmembrane compound described herein, for example, compounds of Formulas Tm-1 to Tm-104.

3.8 T1R1 Transmembrane Compound Binding Site

The present application provides for compositions that modulate the activity of an umami receptor, for example, a T1R1/T1R3 receptor, wherein the compositions interact with one or more amino acids in a transmembrane domain of the umami receptor, for example, a seven transmembrane domain (7TM) in T1R1. In certain embodiments, the amino acids that the compositions interact with comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of Ala795, Ala796, Asn792, Trp773, Phe776, Ala731, Phe728, Leu730, Phe732, Asn735, Ala689, Ser686, Gln690, Ile693, Cys694, Leu695, Arg634, Gln635, Phe642, Ala639, Ala643, and Leu638.

In a specific non-limiting embodiment, the amino acids that the compositions interact with comprise Asn735 and/or Ser686.

In other non-limiting embodiments, the amino acids that the compositions interact with comprise 1, 2, 3, 4, 5, 6, 7 or more of Trp773, Phe776, Phe732, Phe728, Leu730, Leu695, Leu638, and Phe642.

In yet other non-limiting embodiments, the amino acids that the compositions interact with comprise 1, 2, 3, 4, or more of Trp773, Phe776, Phe732, Phe728, and Phe642. In a non-limiting example, the amino acids that interact with the composition undergo ring stacking interactions to the bound composition.

In certain embodiments, the composition interacts with the umami 7TM domain according to any combination of interactions described herein, for example, one, two, three or more of the interactions. The interactions between the composition and the 7TM may further include additional hydrophobic interactions that add to the interaction energy of the composition to the 7TM.

In certain embodiments, the interaction between a composition and the one or more T1R1 7TM domain amino acids comprises one or more hydrogen bond, covalent bond, non-covalent bond, salt bridge, physical interaction, and combinations thereof. The interactions can also be any interaction characteristic of a ligand receptor interaction known in the art. Such interactions can be determined by, for example, site directed mutagenesis, x-ray crystallography, x-ray or other spectroscopic methods, Nuclear Magnetic Resonance (NMR), cross-linking assessment, mass spectroscopy or electrophoresis, cryo-microscopy, displacement assays based on known agonists, structural determination and combinations thereof. In certain embodiments, the interactions are determined in silico, for example, by theoretical means such as docking a compound into the T1R1 7TM domain using molecular docking, molecular modeling, molecular simulation, or other means known to persons of ordinary skill in the art.

The present application also provides for methods of identifying a compound that modulates the activity of an umami receptor, for example, a T1R1, wherein the compound is identified based on its ability to interact with one or more of the amino acids described herein that are present in the 7TM domain of T1R1.

In certain embodiments the method comprises contacting a test agent with a feline T1R1 umami receptor, detecting an interaction between the test agent and one more amino acids in a 7TM interacting site of the feline T1R1 umami receptor, and selecting as the compound, a test agent that interacts with one or more of the amino acids.

4. FLAVOR COMPOSITIONS

In certain embodiments, the flavor compositions of the present disclosure can be used to enhance the umami taste and/or increase the palatability of pet food products, such as cat food products. The flavor compositions can include combinations of compounds, and can be added to the pet food product in various delivery systems.

In certain embodiments, the present disclosure relates to methods for modulating the umami taste of a pet food product comprising: a) providing at least one pet food product, or a precursor thereof, and b) combining the pet food product, or precursor thereof, with at least a umami taste modulating amount of at least one flavor composition, for example, comprising one or more nucleotide derivatives and/or one or more transmembrane compound, or a comestibly acceptable salt thereof, so as to form an enhanced pet food product.

In certain embodiments, the flavor compositions of the present disclosure can enhance the umami taste and/or palatability of a pet food product, such as, for example, a pet food product including wet pet food products, dry pet food products, moist pet food products, pet beverage products and/or snack pet food products.

In certain embodiments, one or more of the flavor compositions of the present disclosure can be added to a pet food product, in an amount effective to modify, enhance or otherwise alter a taste or taste profile of the pet food product. The modification can include, for example, an increase or enhancement in the umami taste of the pet food product, as determined by animals, e.g., cats and/or dogs, or in the case of formulation testing, as determined by a panel of animal taste testers, e.g., cats and/or dogs, via procedures known in the art.

In certain embodiments of the present disclosure, a pet food product can be produced that contains a sufficient amount of at least one flavor composition described herein, for example, comprising a nucleotide derivative, e.g., a compound of Formula Nt-1, to produce a pet food product having the desired taste, e.g., umami.

In certain embodiments of the present disclosure, a pet food product can be produced that contains a sufficient amount of at least one flavor composition described herein, for example, comprising a transmembrane compound, e.g., a compound of Formula Tm-1 to Tm-19, to produce a pet food product having the desired taste, e.g., umami.

In certain embodiments of the present disclosure, a pet food product can be produced that contains a sufficient amount of a flavor composition comprising at least one, two, three, four, five, six or more nucleotide derivatives.

In certain embodiments of the present disclosure, a pet food product can be produced that contains a sufficient amount of a flavor composition comprising at least one, two, three, four, five, six or more transmembrane compounds.

In certain embodiments, a modulating amount of one or more of the flavor compositions of the present disclosure can be added to the pet food product, so that the pet food product has an increased palatability as compared to a pet food product prepared without the flavor composition, as determined by animals, e.g., cats and/or dogs, or in the case of formulation testing, as determined by a panel of animal taste testers, via procedures known in the art.

In certain embodiments of the present disclosure, the flavor composition is added to a pet food product in an amount effective to increase, enhance and/or modify the palatability of the pet food product.

The concentration of flavor composition admixed with a pet food product to modulate and/or improve the palatability and/or umami taste of the pet food product can vary depending on variables, such as, for example, the specific type of pet food product, what umami compounds are already present in the pet food product and the concentrations thereof, and the enhancer effect of the particular flavor composition on such umami compounds.

A broad range of concentrations of the flavor compositions can be employed to provide such umami taste and/or palatability modification. In certain embodiments of the present application, the flavor composition is admixed with a pet food product wherein the flavor composition is present in an amount of from about 0.001 ppm to about 1,000 ppm. For example, but not by way of limitation, the flavor composition can be present in the amount from about 0.001 ppm to about 750 ppm, from about 0.001 ppm to about 500 ppm, from about 0.001 ppm to about 250 ppm, from about 0.001 ppm to about 150 ppm, from about 0.001 ppm to about 100 ppm, from about 0.001 ppm to about 75 ppm, from about 0.001 ppm to about 50 ppm, from about 0.001 ppm to about 25 ppm, from about 0.001 ppm to about 15 ppm, from about 0.001 ppm to about 10 ppm, from about 0.001 ppm to about 5 ppm, from about 0.001 ppm to about 4 ppm, from about 0.001 ppm to about 3 ppm, from about 0.001 ppm to about 2 ppm, from about 0.001 ppm to about 1 ppm, from about 0.01 ppm to about 1,000 ppm, from about 0.1 ppm to 1,000 ppm, from about 1 ppm to 1,000 ppm, from about 2 ppm to about 1,000 ppm, from about 3 ppm to about 1,000 ppm, from about 4 ppm to about 1,000 ppm, from about 5 ppm to about 1,000 ppm, from about 10 ppm to about 1,000 ppm, from about 15 ppm to about 1,000 ppm, from about 25 ppm to about 1,000 ppm, from about 50 ppm to about 1,000 ppm, from about 75 ppm to about 1,000 ppm, from about 100 ppm to about 1,000 ppm, from about 150 ppm to about 1,000 ppm, from about 250 ppm to about 1,000 ppm, from about 250 ppm to about 1,000 ppm, from about 500 ppm to about 1,000 ppm or from about 750 ppm to about 1,000 ppm, and values in between.

In certain embodiments, the flavor composition is present in the pet food product at an amount greater than about 0.001 ppm, greater than about 0.01 ppm, greater than about 0.1 ppm, greater than about 1 ppm, greater than about 2 ppm, greater than about 3 ppm, greater than about 4 ppm, greater than about 5 ppm, greater than about 10 ppm, greater than about 25 ppm, greater than about 50 ppm, greater than about 75 ppm, greater than about 100 ppm, greater than about 250 ppm, greater than about 500 ppm, greater than about 750 ppm or greater than about 1000 ppm, and values in between.

In certain embodiments, a nucleotide derivative of the present disclosure is present in a food product in an amount that is sufficient to modulate, activate and/or enhance an umami receptor, e.g., a T1R1/T1R3 receptor. For example, but not by way of limitation, a nucleotide derivative can be present in a food product in an amount from about 1 pM to about 1 M, from about 1 nM to about 1 M, from about 1 M to about 1 M, from about 1 mM to about 1 M, from about 10 mM to about 1 M, from about 100 mM to about 1 M, from about 250 mM to about 1 M, from about 500 mM to about 1 M, from about 750 mM to about 1 M, from about 0.001 μM to about 1 M, from about 0.001 μM to about 750 mM, from about 0.001 μM to about 500 mM, from about 0.001 μM to about 250 mM, from about 0.001 μM to about 100 mM, from about 0.001 μM to about 50 mM, from about 0.001 μM to about 25 mM, from about 0.001 μM to about 10 mM, from about 0.001 μM to about 1 mM, from about 0.001 M to about 100 μM or from about 0.001 μM to about 10 μM, and values in between.

In certain embodiments, a transmembrane compound of the present disclosure is present in amount of a compound that is sufficient to modulate, activate or enhance an umami receptor, e.g., feline T1R1/T1R3 receptor. For example, but not by way of limitation, a transmembrane compound can be present in a food product in an amount from about 1 pM to about 10 M, from about 1 pM to about 1 M, from about 1 nM to about 1 M, from about 1 μM to about 1 M, from about 1 mM to about 1 M, from about 10 mM to about 1 M, from about 100 mM to about 1 M, from about 250 mM to about 1 M, from about 500 mM to about 1 M, from about 750 mM to about 1 M, from about 1 μM to about 1 M, from about 1 μM to about 750 mM, from about 1 μM to about 500 mM, from about 1 μM to about 250 mM, from about 1 μM to about 100 mM, from about 1 μM to about 50 mM, from about 1 μM to about 25 mM, from about 1 μM to about 10 mM, from about 1 μM to about 1 mM, from about 1 μM to about 100 μM or from about 1 μM to about 10 μM, and values in between.

In certain embodiments of the present application, the flavor composition is admixed with a food product wherein the flavor composition is present in an amount of from about 0.0001 to about 10% weight/weight (w/w) of the food product. For example, but not by way of limitation, the flavor composition can be present in the amount from about 0.0001% to about 10%, from about 0.0001% to about 1%, from about 0.0001% to about 0.1%, from about 0.0001 to about 0.01%, from about 0.0001% to about 0.001%, from about 0.001% to about 10%, from about 0.001% to about 1%, from about 0.01% to about 1% or from about 0.1% to about 1%, and values in between.

In certain embodiments, the nucleotide derivatives and/or transmembrane compounds of the present application are blended together in various ratios or are blended together with other compounds, e.g., nucleotides and/or amino acids and/or furanones, to form various flavor compositions. Non-limiting examples of nucleotides, amino acids and furanones are disclosed in Patent Application Nos. PCT/EP2013/072788, PCT/EP2013/072789, PCT/EP2013/072790, and PCT/EP2013/072794, which are incorporated herein by reference in their entireties.

In certain embodiments, the nucleotide derivatives and/or transmembrane compounds that are blended with other compounds are one or more compounds of Formula Nt-1 to Nt-55 and/or one or more compounds of Formula Tm-1 to Tm-104.

In certain embodiments, the nucleotide derivatives that are blended with other compounds include nucleotide derivatives of Formulas Nt-1 to Nt-55 and Tables 2 and 6-14.

In certain embodiments, the transmembrane compounds that are blended with other compounds include transmembrane compounds of Formulas Tm-1 to Tm-104.

4.1 Nucleotides

In certain embodiments of the present disclosure, the flavor composition comprises at least one nucleotide derivative and/or at least one transmembrane compound, and at least one nucleotide as described herein.

In certain embodiments of the present disclosure, the flavor composition comprises at least two, three, four, five or more nucleotides as described herein. Non-limiting examples of nucleotides include guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), inosine monophosphate (IMP), inosine diphosphate (IDP, inosine triphosphate (ITP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), and xanthosine monophosphate (XMP), xanthosine diphosphate (XDP), and xanthosine triphosphate (XTP), or any nucleotide derivative as described by Formula Nt-1.

In certain embodiments, the flavor composition can include a nucleotide present in a food product which can be present in an amount of from about 1 pM to about 1 M, from about 1 nM to about 1 M, from about 1 μM to about 1 M, from about 1 mM to about 1 M, from about 10 mM to about 1 M, from about 100 mM to about 1 M, from about 250 mM to about 1 M, from about 500 mM to about 1 M, from about 750 mM to about 1 M, from about 1 μM to about 1 M, from about 1 μM to about 750 mM, from about 1 μM to about 500 mM, from about 1 μM to about 250 mM, from about 1 μM to about 100 mM, from about 1 μM to about 50 mM, from about 1 μM to about 25 mM, from about 1 μM to about 10 mM, from about 1 μM to about 1 mM, from about 1 μM to about 100 μM or from about 1 μM to about 10 μM, and values in between.

In certain embodiments, the nucleotide can be present in an amount of greater than about 1 mM or greater than about 2.5 mM of the pet food product. In certain non-limiting embodiments, the nucleotide and/or nucleotide derivative can be present in an amount of less than about 100 mM, less than about 50 mM, less than about 20 mM or less than about 10 mM of the pet food product. In a certain, non-limiting embodiments, the nucleotide is present in an amount of about 5 mM of the pet food product.

In certain embodiments, the flavor composition comprises at least one transmembrane compound and at least one nucleotide and/or nucleotide derivative, which can be IMP, GMP or a mixture thereof. In certain embodiments, the at least one nucleotide can be a combination of GMP and IMP comprising about 1% to about 99% of GMP and from about 1% to about 99% of IMP, or from about 20% to about 80% of GMP and from about 20% to about 80% of IMP, or about 50% GMP and about 50% IMP, or about 10% GMP and about 90% IMP, or about 20% GMP and about 80% IMP, or about 30% GMP and about 70% IMP, or about 40% GMP and about 60% IMP, or about 60% GMP and about 40% IMP, or about 70% GMP and about 30% IMP, or about 80% GMP and about 20% IMP or about 10% GMP and about 90% IMP.

In certain embodiments of the present disclosure, the flavor composition further comprises at least one amino acid as described herein.

4.2 Amino Acids

In certain embodiments of the present disclosure, the flavor composition comprises at least one nucleotide derivative and/or at least one transmembrane compound, and at least one amino acid as described herein. In certain embodiments, the flavor composition comprises at least two, three, four, five or more amino acids as described herein.

In certain embodiments, the flavor composition comprises at least one, two, three, four, five or more first amino acids and/or at least one, two, three, four, five or more second amino acids.

In certain embodiments of the present disclosure, the flavor composition comprises at least one first amino acid and at least one second amino acid.

In certain embodiments of the present disclosure, the flavor composition comprises at least two first amino acids and at least one second amino acid.

In certain embodiments of the present disclosure, the flavor composition comprises at least one first amino acid and at least two second amino acids.

In certain embodiments of the present disclosure, the flavor composition comprises at least two first amino acids and at least two second amino acids.

In certain embodiments of the present disclosure, the flavor composition further comprises at least one nucleotide as described herein.

Non-limiting examples of a first amino acid include tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine and combinations thereof.

Non-limiting examples of a second amino acid include asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxyl proline, arginine, cystine, glutamine, lysine, valine, omithine, taurine, monosodium glutamate (MSG) and combinations thereof.

In certain embodiments, the at least one first amino acid and/or the second amino acid, alone or in combination, can be present in an amount of from about 1 mM to about 1 M, or from about 250 mM to about 1 M, or from about 5 mM to about 500 mM, or from about 10 mM to about 100 mM, or from about 15 mM to about 50 mM, or from about 20 mM to about 40 mM of the pet food product. In certain embodiments, the amino acid(s) can be present at an amount less than about 1 M, less than about 200 mM, less than about 100 mM, less than about 50 mM, less than about 20 mM or less than about 10 mM of the pet food product. In certain embodiments, the first amino acid and/or the second amino acid, alone or in combination, can be present in an amount of about 25 mM of the pet food product.

4.2.1 T1R1 Amino Acid Binding Site

The amino acids of the compositions described herein that modulate an umami receptor, for example, a T1R1/T1R3 receptor, can interact with one or more amino acids in a Venus Flytrap domain of the umami receptor. In certain embodiments, the Venus Flytrap domain (VFT) is present in T1R1. In certain embodiments, the VFT amino acids that the composition interacts with comprises one or more of Thr149, Tyr220, Thr148, Thr449, Ser172, Glu170, Glu301, His71, His47, Arg277, His308, Asn69, Asn302, Ser306, Ser384, Asp302, Ser306, and Ala380.

In one non-limiting embodiment, the composition comprises an amino acid, wherein the amino acid interacts with one, two, three, four, five, six or more of Ser172, Thr149, Thr148, Glu301, Tyr220, Glu170 and Asp302 of T1R1.

In other non-limiting embodiments, the composition interacts with one, two, three, four, five or more of Thr149, Ser172, Tyr220, Thr148, Glu170, and/or Asp302, wherein the interactions can comprise, for example, hydrogen bonds, salt-bridges, and/or Pi-cation interactions.

In a non-limiting example, the composition interacts with Glu170 and/or Asp302 of the VFT domain, wherein the composition does not include L-Glutamate or L-Aspartic Acid. In one embodiment, Glu170 and Asp302 help coordinate the zwitterionic nitrogen of the amino acid ligand of the composition that fits the active site of T1R1, while simultaneously establishing an electrostatic environment that is inhospitable to binding L-Glutamate and L-Aspartic Acid.

In certain embodiments, the composition interacts with the VFT according to any combination of interactions described herein, for example, one, two, three or more of the interactions. The interactions between amino acid and the VFT may further include additional hydrophobic interactions that add to the interaction energy of the amino acid to the VFT.

In certain embodiments, the interaction between the composition and the one or more VFT amino acids comprises one or more hydrogen bond, covalent bond, non-covalent bond, salt bridge, physical interaction, and combinations thereof. The interactions can also be any interaction characteristic of a ligand receptor interaction known in the art. Such interactions can be determined by, for example, site directed mutagenesis, x-ray crystallography, x-ray or other spectroscopic methods, Nuclear Magnetic Resonance (NMR), cross-linking assessment, mass spectroscopy or electrophoresis, displacement assays based on known agonists, structural determination and combinations thereof. In certain embodiments, the interactions are determined in silico, for example, by theoretical means such as docking a compound into the VFT domain using molecular docking, molecular modeling, molecular simulation, or other means known to persons of ordinary skill in the art.

The present application also provides for methods of identifying a compound that modulates the activity of an umami receptor, for example, a T1R1, wherein the compound is identified based on its ability to interact with one or more of the amino acids described herein that are present in the VFT domain of T1R1.

In certain embodiments the method comprises contacting a test agent with a feline T1R1 umami receptor, detecting an interaction between the test agent and one more amino acids in a VFT interacting site of the feline T1R1 umami receptor, and selecting as the compound, a test agent that interacts with one or more of the amino acids.

5. DELIVERY SYSTEMS

In certain embodiments, the flavor compositions of the present application can be incorporated into a delivery system for use in pet food products. Delivery systems can be liquid or solid, aqueous or non-aqueous. Delivery systems are generally adapted to suit the needs of the flavor composition and/or the pet food product into which the flavor composition will be incorporated.

The flavoring compositions can be employed in liquid form, dried form and/or solid form. When used in dried form, suitable drying means such as spray drying can be used. Alternatively, a flavoring composition can be encapsulated or absorbed onto water soluble materials, including but not limited to materials such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth. The actual techniques for preparing such dried forms are well-known in the art, and can be applied to the presently disclosed subject matter.

The flavor compositions of the presently disclosed subject matter can be used in many distinct physical forms well known in the art to provide an initial burst of taste, flavor and/or texture; and/or a prolonged sensation of taste, flavor and/or texture. Without being limited thereto, such physical forms include free forms, such as spray dried, powdered, and beaded forms, and encapsulated forms, and mixtures thereof.

In certain embodiments, the nucleotide derivatives and/or transmembrane compounds of a flavor composition can be generated during processing of the pet food. For example, and not by way of limitation, the nucleotide derivatives and/or transmembrane compounds can be generated from precursor compounds during thermal processing, e.g., retorting, extrusion and/or sterilization, of the pet food.

In certain embodiments, as noted above, encapsulation techniques can be used to modify the flavor systems. In certain embodiments, flavor compounds, flavor components or the entire flavor composition can be fully or partially encapsulated. Encapsulating materials and/or techniques can be selected to determine the type of modification of the flavor system.

In certain embodiments, the encapsulating materials and/or techniques are selected to improve the stability of the flavor compounds, flavor components or flavor compositions; while in other embodiments the encapsulating materials and/or techniques are selected to modify the release profile of the flavor compositions.

Suitable encapsulating materials can include, but are not limited to, hydrocolloids such as alginates, pectins, agars, guar gums, celluloses, and the like, proteins, polyvinyl acetate, polyethylene, crosslinked polyvinyl pyrrolidone, polymethylmethacrylate, polylactidacid, polyhydroxyalkanoates, ethylcellulose, polyvinyl acetatephthalate, polyethylene glycol esters, methacrylicacid-co-methylmethacrylate, ethylene-vinylacetate (EVA) copolymer, and the like, and combinations thereof. Suitable encapsulating techniques can include, but are not limited to, spray coating, spray drying, spray chilling, absorption, adsorption, inclusion complexing (e.g., creating a flavor/cyclodextrin complex), coacervation, fluidized bed coating or other process can be used to encapsulate an ingredient with an encapsulating material.

Encapsulated delivery systems for flavoring agents or sweetening agents can contain a hydrophobic matrix of fat or wax surrounding a sweetening agent or flavoring agent core. The fats can be selected from any number of conventional materials such as fatty acids, glycerides or poly glycerol esters, sorbitol esters, and mixtures thereof. Examples of fatty acids include but are not limited to hydrogenated and partially hydrogenated vegetable oils such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil, safflower oil and combinations thereof. Examples of glycerides include, but are not limited to, monoglycerides, diglycerides and triglycerides.

Waxes can be chosen from the group consisting of natural and synthetic waxes and mixtures thereof. Non-limiting examples include paraffin wax, petrolatum, carbowax, microcrystalline wax, beeswax, camauba wax, candellila wax, lanolin, bayberry wax, sugarcane wax, spermaceti wax, rice bran wax, and mixtures thereof.

The fats and waxes can be use individually or in combination in amounts varying from about 10 to about 70%, and alternatively in amounts from about 30 to about 60%, by weight of the encapsulated system. When used in combination, the fat and wax can be present in a ratio from about 70:10 to 85:15, respectively.

Typical encapsulated flavor compositions, flavoring agent or sweetening agent delivery systems are disclosed in U.S. Pat. Nos. 4,597,970 and 4,722,845, the disclosures of which are incorporated herein by reference in their entireties.

Liquid delivery systems can include, but are not limited to, systems with a dispersion of the flavor compositions of the present application, such as in carbohydrate syrups and/or emulsions. Liquid delivery systems can also include extracts where the nucleotide derivative, transmembrane compound, and/or the flavor compositions are solubilized in a solvent. Solid delivery systems can be created by spray drying, spray coating, spray chilling, fluidized bed drying, absorption, adsorption, coacervation, complexation, or any other standard technique. In some embodiments, the delivery system can be selected to be compatible with or to function in the edible composition. In certain embodiments, the delivery system will include an oleaginous material such as a fat or oil. In certain embodiments, the delivery system will include a confectionery fat such as cocoa butter, a cocoa butter replacer, a cocoa butter substitute, or a cocoa butter equivalent.

When used in dried form, suitable drying means such as spray drying may be used. Alternatively, a flavoring composition may be adsorbed or absorbed onto substrates such as water soluble materials, such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth may be encapsulated. The actual techniques for preparing such dried forms are well known in the art.

6. PET FOOD PRODUCTS

The flavor compositions of the present disclosed subject matter can be used in a wide variety of pet food products. Non-limiting examples of suitable pet food products include wet food products, dry food products, moist food products, pet food supplements (e.g., vitamins), pet beverage products, snack and treats and pet food categories described herein.

The combination of the flavoring composition(s) of the presently disclosed subject matter together with a pet food product and optional ingredients, when desired, provides a flavoring agent that possesses unexpected taste and imparts, for example, an umami and/or savory sensory experience. The flavor compositions disclosed herein can be added prior to, during or after formulation processing or packaging of the pet food product, and the components of the flavor composition can be added sequentially or simultaneously. In certain embodiments, one or more of the components of the flavor compositions disclosed herein can be generated during the production of the pet food product from precursor compounds, e.g., during thermal food processing. For example, and not by way of limitation, a nucleotide derivative and/or transmembrane compound of a flavor composition can be generated during the production of a pet food product and additional components of the flavor composition can be added prior to, during or after formulation processing or packaging of the pet food product.

In certain embodiments, the pet food product is a nutritionally complete dry food product. A dry or low moisture-containing nutritionally-complete pet food product can comprise less than about 15% moisture, and include from about 10 to about 60% fat, from about 10% to about 70% protein and from about 30% to about 80% carbohydrates, e.g., dietary fiber and ash.

In certain embodiments, the pet food product is a nutritionally complete wet food product. A wet or high moisture-containing nutritionally-complete pet food product can comprise greater than about 50% moisture. In certain embodiments, the wet pet food product includes from about 40% fat, from about 50% protein and from about 10% carbohydrates, e.g., dietary fiber and ash.

In certain embodiments, the pet food product is a nutritionally complete moist food product. A moist, e.g., semi-moist or semi-dry or soft dry or soft moist or intermediate or medium moisture containing nutritionally-complete pet food product comprises from about 15 to about 50% moisture.

In certain embodiments, the pet food product is a pet food snack product. Non-limiting examples of pet food snack products include snack bars, pet chews, crunchy treats, cereal bars, snacks, biscuits and sweet products.

In certain embodiments, the protein source can be derived from a plant source, such as lupin protein, wheat protein, soy protein and combinations thereof.

Alternatively or additionally, the protein source can be derived from a variety of animal sources. Non-limiting examples of animal protein include beef, pork, poultry, lamb, or fish including, for example, muscle meat, meat byproduct, meat meal or fish meal.

7. METHODS OF MEASURING TASTE ATTRIBUTES

In certain embodiments of the present disclosure, the taste, flavor and/or palatability attributes of a pet food product can be modified by admixing a flavor composition with the food product, or generated under food preparation conditions, as described herein. In certain embodiments, the attribute(s) can be enhanced or reduced by increasing or decreasing the concentration of the flavor composition admixed or generated with the food product. In certain embodiments, the taste attributes of the modified food product can be evaluated as described herein, and the concentration of flavor composition admixed or generated with the food product can be increased or decreased based on the results of the evaluation.

In certain embodiments of the present disclosure, the taste and/or palatability attributes can be measured using an in vitro assay, wherein a compound's ability to activate a feline umami receptor expressed by cells in vitro at different concentrations is measured. In certain embodiments, an increase in the activation of the receptor correlates with an increase in the taste and/or palatability attributes of the compound. In certain embodiments, the composition is measured alone or in combination with other compounds. In certain embodiments the in vitro assay comprises the in vitro assays described in the Examples section of the present application. In some embodiments, the in vitro assay comprises recombinant cells expressing an umami receptor encoded by nucleic acid introduced into the cells (e.g., an exogenous nucleic acid). In other non-limiting embodiments, the in vitro assay comprises cells expressing an umami receptor that is native to the cells. Examples of such cells expressing a native umami receptor include, for example but not limited to, cat and/or dog taste cells. In certain embodiments, the cat and/or dog taste cells expressing an umami receptor are isolated from a cat and/or dog and cultured in vitro.

In certain embodiments of the present disclosure, the taste and/or palatability attributes can be measured using a panelist of taste testers. For example, but not by way of limitation, the panel can contain feline panelists. In certain embodiments, the panel can include canine panelists. In certain embodiments, the palatability of a pet food product can be determined by the consumption of a pet food product containing a flavor composition alone (e.g., the one bowl test, monadic ranking). In certain embodiments, the palatability of a pet food product can be determined by the preferential consumption of a pet food product containing a flavor composition, disclosed herein, versus a pet food product that does not contain the flavor composition or another flavor composition (e.g., the two bowl test for testing preference, difference and/or choice).

In certain embodiments, the palatability and/or umami taste of a flavor composition can be determined by the preferential consumption of a water solution containing a flavor composition, disclosed herein, versus a water solution that does not contain the flavor composition or contains a different flavor composition (e.g., the two bottle test). For example, a solution panel can be used to compare the palatability of a range of concentrations of compounds in a monadic exposure. In certain embodiments, the solution can contain a palatability enhancer, for example, L-histidine, as an ingestive/positive tastant to increase baseline solution intake, therefore enabling the identification of a potential negative impact of the test compound.

The intake ratio for each pet food product or water solution can be determined by measuring the amount of one ration consumed divided by the total consumption. The consumption ratio (CR) can then be calculated to compare the consumption of one ration in terms of the other ration to determine the preferential consumption of one food product or water solution over the other. Alternatively or additionally, the difference in intake (g) can be used to assess the average difference in intake between the two solutions in a two bottle test or between two pet food products in a two bowl test at a selected significance level, for example, at the 5% significance level to determine an average difference in intake with a 95% confidence interval. However, any significance level may be used, for example, a 1, 2, 3, 4, 5, 10, 15, 20, 25, or 50% significance level. In certain embodiments, percentage preference scores, e.g., the percentage preference for one solution or food product by an animal is the percentage of the total liquid or food product ingested during the test that that solution or food product accounts for, can also be calculated.

8. METHODS OF GENERATION

In certain embodiments, the nucleotide derivatives and/or transmembrane compounds of the present disclosure can be generated using standard chemosynthesis processes. In certain embodiments, the chemosynthesis process provides a nucleotide derivative and/or transmembrane compound having a purity of at least 99.999%, or at least 99%, or at least 95%, or at least 90%, or at least 85 or at least 80%. In certain embodiments, the nucleotide derivatives and/or transmembrane compounds can be prepared using standard hydrolysis processes such as those employing acids, enzymes or a combination of acids and enzymes.

The nucleotide derivatives and/or transmembrane compounds of the present disclosure can also be generated under food preparation conditions, e.g., during the production of a pet food product. For example, but not by way of limitation, the nucleotide derivatives and/or transmembrane compounds of the present disclosure can be generated during a thermal food process, e.g., sterilization, retorting and/or extrusion, from precursor compounds present in the pet food. In certain embodiments, a liquid and/or a powder palatant can also be added to enhance the taste of a pet food, e.g., to a dry pet food product, and to increase the palatability of the pet food. The palatant can be a digest of meat (e.g., liver) and/or a digest of a vegetable, and can optionally include other palatants known in the art. In certain embodiments, the nucleotide derivative and/or transmembrane compound can be admixed with or generated in the liquid and/or powder palatant prior to its addition to the pet food product. Alternatively or additionally, the nucleotide derivative and/or transmembrane compound can be admixed with or generated in the liquid and/or powder palatant after its addition to the pet food product.

In certain embodiments, the flavor compositions of the present disclosure comprise one or more transmembrane compounds of Formulas Tm-1 to Tm-104. In certain embodiments, such compounds can, without limitation, be synthesized by any means known in the art. In certain embodiments, parabanic acid derivative transmembrane compound compounds can be synthesized according to the following synthesis scheme:

9. NON-LIMITING EXAMPLES OF FLAVOR COMPOSITIONS OF THE PRESENT DISCLOSURE

As described herein, there are at least three different binding sites present on the feline T1R1/T1R3 receptor (i.e., umami receptor) that allows for binding of small molecules and/or compounds. One of the binding sites present on the feline T1R1/T1R3 receptor can bind nucleotide and/or nucleotide derivatives as described herein. A second binding site present on the feline T1R1/T1R3 receptor can bind first group amino acids as described herein, and a third binding site present on the feline T1R1/T1R3 receptor (i.e., the T1R1 7TM domain) can bind transmembrane compounds as described herein. Without being bound to any particular theory, the binding of a first group amino acid, as disclosed herein, can change the conformation of the feline T1R1/T1R3 receptor to allow greater contact with a bound nucleotide and/or nucleotide derivative, and result in synergistic activation of the umami receptor. The second group amino acids, disclosed herein, can interact with one or more other receptors and do not compete with first amino acids for binding to the umami receptor. The addition of a second group amino acid to a flavor composition can enhance the flavor perception of the composition. Binding of a transmembrane compound to the receptor, as described herein, further activates the receptor, thereby enhancing or modifying the palatability of a food product comprising such compounds.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide and/or nucleotide derivative that binds to a first binding site on the umami receptor and/or at least one first group amino acid that binds to a second binding site on the umami receptor and/or at least one transmembrane compound that binds to a third binding site on the umami receptor (e.g. within the 7TM domain of an umami receptor) and/or at least one second group amino acid that binds to a distinct receptor.

The presently disclosed subject provides flavor compositions that comprise at least one, two, three, four, five or more nucleotide derivatives and/or at least one, two, three, four, five or more nucleotides and/or at least one, two, three, four, five or more transmembrane compounds and/or at least one, two, three, four, five or more first group amino acids and/or at least one, two, three, four, five or more second group amino acids.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, and at least one, two, three, four, five or more first group amino acids and/or at least one, two, three, four, five or more second group amino acids selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative and at least one first group amino acid selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative and at least one second group amino acid selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, and at least one first group amino acid and at least one second group amino acid selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, and at least two first group amino acids and at least one second group amino acid selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, and at least one first group amino acid and at least two second group amino acids selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, and at least two first group amino acids and at least two second group amino acids selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, and at least one nucleotide and at least one first group amino acid selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, and at least one nucleotide and at least one second group amino acid selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, and at least one nucleotide and at least one, two, three, four, five or more first group amino acids and/or at least one, two, three, four, five or more second group amino acids selected from Table 4.

In certain embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, at least one nucleotide, and at least one first group amino acid and at least one second group amino acid selected from Table 4.

TABLE 4 Amino acids. First Group amino acids: Second Group amino acids: L-Tryptophan L-Proline L-Phenylalanine Hydroxy-L-proline L-Histidine L-Glutamic acid Glycine Monosodium glutamate (MSG) L-Cysteine L-Aspartic acid L-Alanine L-Arginine L-Tyrosine L-Cystine L-Serine L-Glutamine L-Methionine L-Isoleucine L-Leucine L-Lysine L-Asparagine L-Threonine L-Valine L-Ornithine

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, histidine and proline.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, alanine and proline.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, glycine and proline.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, phenylalanine and proline.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, tryptophan and proline.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, tyrosine and proline.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, histidine and threonine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, alanine and threonine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, glycine and threonine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, phenylalanine and threonine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, tryptophan and threonine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, tyrosine and threonine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, histidine and glutamic acid.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, alanine and glutamic acid.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, glycine and glutamic acid.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, phenylalanine and glutamic acid.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, tryptophan and glutamic acid.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising at least one nucleotide derivative, tyrosine and glutamic acid.

In certain embodiments, any of the flavor compositions disclosed above can further comprise at least one nucleotide and/or at least one transmembrane compound, as described herein.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising adenosine 3′,5′-diphosphate and alanine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising 6-thioguanosine-5′-O-monophosphate and alanine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising 2′-, 3′-O—(N′-methylanthraniloyl)guanosine-5′-O-monophosphate and alanine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising 2-amino-6-chloropurineriboside-5′-O-monophosphate and alanine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising 6-chloropurineriboside-5′-O-monophosphate and alanine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising inosine triphosphate (ITP) and alanine.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising inosine triphosphate (ITP), alanine and IMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising 1-(2-bromophenyl)-3-((1R, 2S)-2-hydroxy-2,3-dihydro-1H-inden-1-yl)urea, alanine and IMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising N-(benzo[d][1,3]dioxol-5-yl)-2-propylpentanamide, alanine and IMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide, alanine and IMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide, alanine and IMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising (E)-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide, alanine and IMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione, alanine and IMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising 1H-imidazo[4,5-c]pyridin-2(3H)-one, alanine and IMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide, phenylalanine and GMP.

In certain non-limiting embodiments, the present disclosure provides for a flavor composition comprising a combination of N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide with a first amino acid (Group A amino acid), second amino acid (Group B amino acid) and one or more nucleotide, as described below.

TABLE 5 Flavor compositions comprising a transmembrane compound, first amino acid (Group A), second amino acid (Group B) and a nucleotide. Group Group A B amino amino Transmembrane compound: acid: acid: Nucleotide: N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Ala Pro IMP + GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Ala Pro IMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Ala Pro GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Gly Pro IMP + GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Gly Pro IMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Gly Pro GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- His Pro IMP + GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- His Pro IMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- His Pro GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Ala Thr IMP + GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Ala Thr IMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Ala Thr GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Gly Thr IMP + GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Gly Thr IMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- Gly Thr GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- His Thr IMP + GMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- His Thr IMP carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- His Thr GMP carboxamide

In certain non-limiting embodiments, the present disclosure provides for a pet food product comprising a flavor composition as described herein, wherein the flavor composition is present in an amount of from about 0.001 ppm to about 1,000 ppm.

In certain non-limiting embodiments, the present disclosure provides for a pet food product comprising a flavor composition as described herein, wherein the flavor composition is present at a concentration of from about 0.0001% weight to about 10% weight of the pet food product.

In certain non-limiting embodiments, the present disclosure provides for a pet food product comprising a flavor composition as described herein, wherein the flavor composition is present in an amount greater than about 1 ppm.

In certain non-limiting embodiments, the present disclosure provides for a pet food product comprising a flavor composition as described herein, wherein the flavor composition is present in an amount greater than about 10 ppm.

In certain non-limiting embodiments, the present disclosure provides for a pet food product comprising a flavor composition as described herein, wherein the flavor composition is present in an amount greater than about 100 ppm.

In certain non-limiting embodiments, the present disclosure provides for a pet food product comprising a nucleotide derivative as described herein, wherein the nucleotide derivative is present in an amount from about 1 pM to about 1 M.

10. NON-LIMITING EXAMPLES OF METHODS OF THE PRESENT DISCLOSURE

In certain non-limiting embodiments, the present disclosure provides for a method of increasing the palatability of a pet food product comprising admixing the pet food product with a flavor composition comprising a nucleotide derivative and/or a transmembrane compound as described herein, wherein the nucleotide derivative and/or transmembrane compound is present at a concentration of from about 1 pM to about 10 M, or from about 1 pM to about 1 M in the admixture.

In certain non-limiting embodiments, the present disclosure provides for a method of increasing the palatability of a pet food product comprising producing the pet food product with a flavor composition comprising a nucleotide derivative and/or a transmembrane compound as described herein, wherein the nucleotide derivative and/or a transmembrane compound is present at a concentration of from about 1 pM to about 10 M, or from about 1 pM to about 1 M in the product.

In certain non-limiting embodiments, the present disclosure provides for a method of increasing the umami taste of a pet food product comprising admixing the pet food product with a flavor composition comprising a nucleotide derivative and/or a transmembrane compound as described herein, wherein the nucleotide derivative is present at a concentration of from 0.001 ppm to 1,000 ppm in the admixture.

In certain non-limiting embodiments, the present disclosure provides for a method of increasing the palatability of a pet food product comprising admixing the pet food product with a flavor composition comprising a nucleotide derivative and/or a transmembrane compound as described herein, wherein the flavor composition is present at a concentration of from about 0.001 ppm to 1,000 ppm in the admixture.

In certain non-limiting embodiments, the present disclosure provides for a method of increasing the umami taste of a pet food product comprising admixing the pet food product with a flavor composition comprising a nucleotide derivative and/or a transmembrane compound as described herein, wherein the flavor composition is present at a concentration of from about 0.0001% to about 10% weight in the admixture.

11. EXAMPLES

The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.

Example 1—Activation of T1R1/T1R3 Receptor by Nucleotide Derivatives

The present example describes the activation of the cat T1R1/T1R3 receptor by nucleotide derivatives in vitro.

Nucleotide derivatives that may function as T1R1/T1R3 activators were identified by in silico modeling with the umami cat receptor, T1R1/T1R3, and selected for further testing in vitro. In vitro functional characterization of the selected nucleotide derivatives was used to evaluate the effectiveness of a nucleotide derivative in activating the T1R1/T1R3 receptor alone and in combination with amino acids and/or nucleotides.

Methods:

HEK293 cells that stably express T1R3 and inducibly express T1R1 were exposed to nucleotide derivatives alone to activate the umami receptor. Activation of the T1R1/T1R3 receptor was detected by a change in intracellular calcium levels using a calcium sensitive fluorescent dye. Cells that express T1R3 but not T1R1 were used as a control. A FLIPR® Tetra or a FlexStation® 3 was used for data capture.

For each nucleotide derivative, dose response curves were generated and the following properties were determined: EC₅₀ of the nucleotide derivative alone; EC₅₀ of the nucleotide derivative with 20 mM alanine; EC₅₀ of the nucleotide derivative with 0.2 mM IMP; and EC₅₀ of the nucleotide derivative with 20 mM alanine and 0.2 mM IMP.

The term half maximal effective concentration (EC₅₀o) refers to the concentration of a compound which induces a response halfway between the baseline and the maximum after a specified exposure time. In each experiment, serial dilutions of up to 0.1 mM, 1 mM or 10 mM of the nucleotide derivative were added to the T1R1/T1R3-expressing cells.

Results:

Treatment of HEK293 cells expressing T1R1/T1R3 receptors with 2′-deoxyadenosine-3′, 5′-O-bisphosphate alone (e.g., in buffer), or in combination with 20 mM alanine, resulted in the activation of the T1R1/T1R3 receptor, as indicated by the change in intracellular calcium levels (ΔF/F₀). In the presence of alanine or in buffer, 2′-deoxyadenosine-3′, 5′-O-bisphosphate resulted in an observed EC₅₀ value of 0.02 mM (Table 6 and FIG. 2). These results indicated that 2′-deoxyadenosine-3′, 5′-O-bisphosphate is a positive activator of the T1R1/T1R3 receptor.

The nucleotide derivative, adenosine 5′-O-thiomonophosphate dilithium salt, was observed to function as an activator of T1R1/T1R3 alone (FIG. 8 and Table 6).

In the presence of 20 mM alanine, which has an EC₅₀ value of 13.6 mM alone, the EC₅₀ of the dilithium salt form of adenosine 5′-O-thiomonophosphate decreased from greater than 1 mM to 0.06 mM and the ΔF/F₀ extended significantly higher (FIG. 8 and Table 6). These results suggest that alanine and nucleotide derivatives, e.g., adenosine 5′-O-thiomonophosphate, act synergistically to activate T1R1/T1R3.

Without being bound to a particular theory, these results show that nucleotide derivatives alone or in combination with an amino acid, e.g., alanine, can function as positive modulators of the T1R1/T1R3 receptor.

TABLE 6 Activation of T1R1/T1R3 by nucleotide derivatives EC₅₀- EC₅₀- EC₅₀- with with EC₅₀- with alone Ala IMP Ala and Name Chemical Structure CAS No. (mM) (mM) (mM) IMP (mM) 2′-Deoxyadenosine- 3′,5′-O- bisphosphate

16174- 58-6 0.02 ± 0.009  0.02 ± 0.005 — — Inosine 5′- disphosphate (IDP) sodium salt

81012- 88-6 — 0.65 ± 0.07 — — 2′-/3′-O-(2- Aminoethyl- carbamoyl) guanosine-5′-O- monophosphate (2′-/ 3′-AEC-5′-GMP)

N/A 0.07 ± 0.04  0.14 ± 0.02 >3 >3 Inosine Triphosphate (ITP) trisodium salt

35908- 31-7 0.05 ± 0.03  0.08 ± 0.01 0.02 ± 0.03 0.03 ± 0.01 N6-Benzoyl- adenosine-5′-O- monophosphate (6-Bnz-5′-AMP)

40871- 55-4 >1 >1 0.72 ± 0.25 >1 Adenosine 5′-O- thiomonophosphate dilithium salt

93839- 85-1 >1  0.06 ± 0.03 >1 >1 Adenosine 3′,5′- diphosphate sodium salt

75431- 54-8 — 0.001 ± 0.1 — — Adenosine 3′,5′- diphosphate sodium salt + Ala (1:1000)

75431- 54-8 >0.01 >0.01 >0.01 — Adenosine 3′,5′- diphosphate sodium salt + Ala (1:100)

75431- 54-8 >0.03 0.003 ± 0.0003 — — Adenosine 3′,5′- diphosphate sodium salt + Ala (1:10)

75431- 54-8 >0.03 0.003 ± 0.0003 — —

Example 2—Activation of T1R1/T1R3 Receptor by Nucleotide Derivative Compounds in Combination with an Amino Acid

The present example describes the activation of the cat T1R1/T1R3 receptor by nucleotide derivatives in combination with an amino acid in vitro.

Nucleotide derivatives were evaluated to determine the effectiveness of the nucleotide derivatives in activating the T1R1/T1R3 receptor in combination with one or more nucleotides and/or one or more amino acids.

Methods:

HEK293 cells that stably express T1R3 and inducibly express T1R1 were exposed to nucleotide derivatives alone or in combination with one or more amino acids and/or one or more nucleotides to activate the umami receptor. Activation of the T1R1/T1R3 receptor was detected by a change in intracellular calcium levels using a calcium sensitive fluorescent dye and/or a luminescent reporter system. Cells that express T1R3 but not T1R1 were used as a control. A FLIPR® Tetra or a FlexStation® 3 was used for data capture.

For each nucleotide derivative, dose response curves were generated and the following properties were determined in the presence of 20 mM alanine: the EC₅₀ of the nucleotide derivative, maximum response of the receptor in the presence of the nucleotide derivative, the maximum response of the receptor in the presence of the nucleotide derivative in relation to the response of the receptor in the presence of IMP and the threshold amount of the nucleotide derivative that results in the activation of the T1R1/T1R3 receptor. EC₅₀ values for the positive and negative controls used are summarized in Table 16.

Results:

The effect of all nucleotide derivatives tested in activating T1R1/T1R3 is shown in table 15. Treatment of HEK293 cells expressing T1R1/T1R3 receptors with the nucleotide derivative, adenosine 3′,5′-diphosphate sodium salt (ADP), in combination with 20 mM alanine resulted in the activation of the T1R1/T1R3 receptor, as indicated by the maximum change in intracellular calcium levels (ΔF/F₀) and the observed EC₅₀ value of 0.001 mM. In contrast, the nucleotide, adenosine monophosphate (AMP), in the presence of 20 mM alanine, resulted in an observed EC₅₀ value of 0.011 mM (Table 7). These results indicate that the adenosine-based nucleotide derivative exhibited improved activity at activating the T1R1/T1R3 receptor compared to the standard nucleotide from which it was derived. Similar results were observed for 2′-/3′-O—(N′-Methyl-anthraniloyl) adenosine-5′-O-monophosphate sodium salt (Table 7). In addition, the adenosine-based nucleotide derivatives also exhibited a lower threshold value for activating the receptors as compared to AMP (Table 7).

The guanosine-based nucleotide derivatives, 6-thioguanosine-5′-O-monophosphate, 2′-deoxyguanosine-5′-O-monophosphorothioate (sodium salt); 2′-, 3′-O—(N′-methylanthraniloyl) guanosine-5′-O-monophosphate; guanosine-5′-monophosphorothioate (sodium salt); 2′-deoxy-3′-O—(N′-methylanthraniloyl) guanosine-5′-O-monophosphate; Guanosine-5′-O-(2-thiodiphosphate), 2′-deoxyguanosine-3′,5′-O-bisphosphate; and 2′-deoxyguanosine-5′-O-monophosphorothioate were observed to function as an activators of T1R1/T1R3 in the presence of alanine (Table 8). As shown in Table 8, these guanosine-based nucleotide derivatives exhibited improved activity as compared to the standard nucleotide, guanosine monophosphate (GMP). For example, 6-thioguanosine-5′-O-monophosphate exhibited an EC₅₀ value of 0.0009 mM in the presence of alanine and a threshold value of 0.0002 mM; whereas, GMP exhibited an EC₅₀ value of 0.02 mM and a threshold value of 0.008 mM (Table 8 and FIG. 1).

Purine-based nucleotide derivatives, 2-Amino-6-chloropurineriboside-5′-O-monophosphate (2-NH2-6-Cl-5′-PuMP) and 6-chloropurineriboside-5′-O-monophosphate also were observed to function as activators of the T1R1/T1R3 receptor (Table 9). For example, 2-Amino-6-chloropurineriboside-5′-O-monophosphate, in the presence of alanine, exhibited an EC₅₀ value of 0.0005 mM with a threshold value of 0.00013 mM, and 6-chloropurineriboside-5′-O-monophosphate, in the presence of alanine, exhibited an EC₅₀ value of 0.002 mM with a threshold value of 0.0005 mM (Table 9). In contrast, purine riboside-5′-O-monophosphate (5′-PuMP) exhibited an EC₅₀ value of 0.02 mM with a threshold value of 0.005 mM in the presence of alanine, indicating that the purine-based nucleotides activated the cat Umami receptor at a lower concentration as compared to the standard nucleotide, 5′-PuMP (Table 9).

Inosine-based nucleotide derivatives were observed to activate the receptor in the presence of alanine (Table 11). 6-thioinosine phosphate exhibited an EC₅₀ value of 0.02 mM in the presence of alanine and inosine triphosphate (ITP) exhibited an EC₅₀ value of 0.08 mM in the presence of alanine (Table 11 and FIG. 6). The standard nucleotide, inosine monophosphate (IMP), exhibited an EC₅₀ value 0.07 mM, indicating that 6-thioinosine phosphate exhibited increased activity as compared to IMP. Similar results were observed for uridine-based nucleotide derivatives, where the nucleotide derivatives exhibited improved activity compared to uridine monophosphate (UMP). For example, the uridine-based nucleotide derivative, uridine 5′-monophosphomorpholidate 4-morpholine-N,N′-dicyclohexylcarboxamidine, exhibited an EC₅₀ value of greater than 3 mM compared to the UMP EC₅₀ value of greater than 30 mM (Table 12).

Without being bound to a particular theory, these results show that nucleotide derivatives function as positive modulators of the T1R1/T1R3 receptor and exhibit improved activity as compared to standard nucleotides.

TABLE 7 Activation of T1R1/T1R3 by adenosine and adenosine-based nucleotide derivatives EC₅₀- Max. with Max resp. Ala resp. % of Threshold Name Chemical Structure CAS No. (mM) (ΔF/F) IMP (mM) Adenosine-3′,5′- diphosphate sodium salt (ADP)

75431-54-8 0.001 0.9578 98.9 0.00025 2′-/3′-O- (N′-Methyl- anthraniloyl) adenosine- 5′-O- monophosphate sodium salt (MANT-AMP)

85287-53-2 0.01 1.915 154.1 0.0025  Adenosine 5′- monophosphate disodium salt (AMP)

4578-31-8 0.011 1.5470 159.8 0.003  2′-Deoxy- 3′-O-(N′- methyl- anthraniloyl) adenosine-5′- O- monophosphate

N/A 0.01 0.3975 32.0 0.0025  Adenosine- 5′-O- monophos- phorothioate (5′-AMPS)

19341-57-2 0.011 1.2435 128.4 0.003  1, N6- Ethenoadenosine- 5′-O- monophosphate (ϵ-5′-AMP)

103213-41-8 0.04 1.4766 152.5 0.010  2′-Deoxy- N⁶-methyl adenosine 3,5′- diphosphate diammonium salt (MRS 2179 ammonium salt hydrate)

101204-49-3 0.05 1.8150 146.1 0.013  2′-Deoxy- adenosine-3′, 5′-O- bisphosphate

16174-58-6 0.02 0.8 76.4 0.005  Adenosine 5′- phosphosulfate sodium salt

102029-95-8 0.03 0.8658 89.4 0.008  N6- Cyclopentyl- adenosine- 5′-O- monophosphate (6-cPe-5′-AMP)

117778-38-8 0.05 1.2317 127.2 0.013  Adenosine 5′-O- thiomonophosphate dilithium salt

93839-85-1 0.06 1.4 139.4 0.015  2′-Deoxy- adenosine-5′-O- monophos- phorothioate, sodium salt (5′-dAMPS)

64145-26-2 0.06 1.0375 83.5 0.015  2′-/3′-O-(2- Aminoethyl carbamoyl) adenosine-5′- O- monophosphate (2′-/ 3′-AEC-5′-AMP)

81012-88-6 0.09 2.2 213.9 0.0225  N6-Methyl- adenosine 5′- monophos- phate sodium salt

81921-35-9 0.08 0.9907 102.3 0.020  2′-Deoxy- adenosine 5′- monophosphate

653-63-4 0.14 1.1503 118.8 0.035  2′-Deoxy-1,N6- etheno- adenosine-5′-O- monophosphate

60508-81-8 0.2 0.87 70.0 0.05   7- Deazaadenosine- 5′-O- monophosphate (7- CH-5′-AMP/ 5′-TuMP)

16719-46-3 0.75 0.4979 51.4 0.188  8- Chloroadenosine- 5′-O- monophosphate, sodium salt 8-Cl-5′-AMPS

37676-40-7 0.8 0.79 63.6 0.2   N6- Benzoyl- adenosine- 5′-O- monophosphate (6-Bnz- 5′-AMP)

40871-55-4 >1 1.1 105.6 0.3  

TABLE 8 Activation of T1R1/T1R3 by guanosine and guanosine-based nucleotide derivatives EC₅₀- Max. with Max resp. Ala resp. % of Threshold Name Chemical Structure CAS No. (mM) (ΔF/F) IMP (mM) 6-Thiogaunosine- 5′-O- monophosphate (6-T-5′-GMP)

74686-78-5 0.0009 1.3614 140.6    0.0002 2′-Deoxyguanosine- 5′-O-mono- phosphorothioate, sodium salt

87358-26-7 0.01  1.0775 86.7    0.0025 2′-,3′-O-(N′- Methylanthraniloyl) guanosine-5′-O- monophosphate (MANT-5′-GMP) Fluorescent analogue of 5′-GMP

85287-54-3 0.0056 0.7190 74.3    0.0014 Guanosine-5′- mono- phosphorothioate sodium salt (5′-GMPS)

76310-16-2 0.01  1.29  103.8    0.0025 2′-Deoxy-3′-O-(N′- methylanthraniloyl) guanosine-5′-O- monophosphate

N/A 0.01  0.295  23.7    0.0025 Guanosine-5′-O-(2- thiodiphosphate)

71376-97-1 0.01  0.5875 47.3    0.0025 2′-Deoxyguanosine- 3′,5′- O-bisphosphate

16174-59-7 0.02  1.8925 152.3   0.005 2′- Deoxyguanosine- 5′-O- monophos- phorothioate (5′-dGMPS)

87358-26-7 0.02  0.8446 87.2   0.005 Guanosine monophosphate (GMP)- disodium salt

5550-12-9 0.03  1.2786 132.1   0.008 N-Lactoyl-5′-GMP

N/A 0.1   1.3600 140.5   0.025 N-Acetyl-5′-GMP

N/A 0.1   1.3351 137.9   0.025 Guanosine 5′- diphosphate sodium salt

43139-22-6 0.2   1.7550 141.2   0.050 2′-/3′-O-(2- Aminoethyl- carbamoyl) guanosine-5′-O- monophosphate (2′-/3′-AEC- 5′-GMP)

N/A 0.14  1.1   109.5   0.035 Guanosine 5′- triphosphate sodium salt hydrate

36051-31-7 0.7   1.8075 145.5   0.175 2′- Deoxyguanosine 5′- monophosphate sodium salt hydrate

N/A 0.24  0.5926 61.2   0.060 Guanosine 5′- monophos- phomorpholidate 4-morpholine- N,N′-dicyclohexyl- carboxamidine salt

7361-07-1 1.3   0.5121 52.9   0.325 8-Bromoguanosine- 5′-O- monophosphate (8-Br-5′-GMP)

21870-09-7 CNBD 0.375  30.2  1 

TABLE 9 Activation of T1R1/T1R3 by purine-based nucleotide derivatives EC₅₀- Max with Ala resp. Max. resp. Threshold Chemical Name Chemical Structure CAS No. (mM) (ΔF/F) % of IMP (mM) 2-Amino-6- chloropurineriboside-5′- O-monophosphate (2-NH2-6-Cl-5′-PuMP)

16321-98-5 0.0005 1.8700 150.5 0.00013 6-Chloropurineriboside- 5′-O-monophosphate, sodium salt

5843-59-4 0.002  1.5325 123.3 0.0005  Purine riboside-5′-O- monophosphate (5′- PuMP)

4578-31-8 0.02  2.0266 209.3 0.005 

TABLE 10 Activation of T1R1/T1R3 by xanthosine EC₅₀- Max with Ala resp. Max. resp. Threshold Chemical Name Chemical Structure CAS No. (mM) (ΔF/F) % of IMP (mM) Xanthosine 5′- Monophosphate Disodium Salt (XMP)

25899-70-1 0.1 1.6223 167.6 0.025

TABLE 11 Activation of T1R1/T1R3 by inosine and inosine-based nucleotide derivatives EC₅₀- Max with Ala resp. Max. resp. Threshold Chemical Name Chemical Structure CAS No. (mM) (ΔF/F) % of IMP (mM) 6-Thioinosine Phosphate

53-83-8 0.02 1.3875 111.7  0.005  Inosine 5′- monophosphate disodium salt (IMP)

352195-40-5 0.07 0.8681 89.7  0.018  Inosine Triphosphate (ITP) trisodium salt

35908-31-7 0.08 0.3   25.1  0.02  2′-Deoxyinosine- 5′-O- monophos- phorothioate, sodium salt

771477-45-3 0.1  1.0375 83.5  0.025  Inosine 5′- disphosphate (IDP) sodium salt

81012-88-6 0.65 0.3   33.3  0.1625

TABLE 12 Activation of T1R1/T1R3 by uridine and uridine-based nucleotide derivatives EC₅₀- Max with Ala resp. Max. resp. Threshold Chemical Name Chemical Structure CAS No. (mM) (ΔF/F) % of IMP (mM) Uridine 5′- monophosphomorph- olidate 4-morpholine- N,N′- dicyclohexyl- carboxamidamine salt

24558-91-6 >3  1.0150 104.8 2.000 2′-Deoxyuridine- 5′-O- monophosphorothioate (5′-dUMPS)

205379-91-5 >30 0.4200  43.4 5.000 Uridine 5′- monophosphate disodium salt (UMP)

3387-36-8 >30 0.2316  23.9 3.000

TABLE 13 Activation of T1R1/T1R3 by cytidine EC₅₀- Max with Ala resp. Max. resp. Threshold Chemical Name Chemical Structure CAS No. (mM) (ΔF/F) % of IMP (mM) Cytidine 5′- monophosphate (CMP)

63-37-6 5.7 0.3066 31.7 1.425

TABLE 14 Activation of T1R1/T1R3 by miscellaneous nucleotide derivatives Max. EC₅₀- Max resp. with Ala resp. % of Threshold Chemical Name Chemical Structure CAS No. (mM) (ΔF/F) IMP (mM) Ribavirin 5′- Mono- phosphate, Dilithium salt

66983-94-6 0.7 1.5294 158.0 0.175 5-Amino- imidazole-4- carboxamide- 1-β-D- ribofuranosyl 5′- monophosphate (AICAR)

3031-94-5 >1  0.6985  72.2 0.200 6- Thioguanosine- 5′-O- disphosphate

16541-19-8 0.18 ± 0.02 1.047  152.8 0.03  6-Chloropurine riboside-5′-O- triphosphate

55673-61-5 >0.03 0.345   50.4 0.01 

TABLE 15 Modulation of T1R1/T1R3 by nucleotide derivatives +Alanine (20 mM) Max Max EC₅₀ resp resp % Thres Compound Name Activity (mM) ΔF/F of IMP (mM) N6-Cyclopentyladenosine-5′-O-monophosphate active 0.05 1.2317 127.2 0.013 (6-cPe-5′-AMP) 8-(4-Chlorophenylthio)adenosine-5′-O-monophosphate (8-pCPT-5′-AMP) unknown N/D N/D N/D N/D 1,N6-Ethenoadenosine-5′-O-monophosphate (ε-5′-AMP) active 0.04 1.4766 152.5 0.010 Guanosine-3′-O-monophosphate (3′-GMP) N/A N/D N/D N/D N/D Guanosine-2′,3′-cyclic monophosphate N/A N/D N/D N/D N/D Purine riboside-5′-O-monophosphate (5′-PuMP) active 0.02 2.0266 209.3 0.005 6-Azauridine-5′-monophosphate unknown N/D N/D N/D N/D 6-Aza-D-uridine-5′-monophosphate 1-(b-D-Arabinofuranosyl)cytosine 5′-monophosphate unknown N/D N/D N/D N/D 5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydro-2-furancarboxylic acid unknown N/D N/D N/D N/D [Di-deoxy thymidine] or SODIUM (5-(5-METHYL-2,4-DIOXO-3,4- N/A N/D N/D N/D N/D DIHYDROPYRIMIDIN-1(2H)-YL)TETRAHYDROFURAN-2-YL)METHYL PHOSPHATE Adenosine 5′-monophosphate disodium salt (AMP) active 0.011 1.5470 159.8 0.003 [AICAR] or 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5′-monophosphate active >1 0.6985 72.2 0.200 8-(6-Aminohexyl)aminoadenosine 5′-monophosphate lithium salt N/A N/D N/D N/D N/D Adenosine 5′-phosphosulfate sodium salt active 0.03 0.8658 89.4 0.008 Adenosine 3′,5′-diphosphate sodium salt active 0.001 0.9578 98.9 0.00025 Cytidine 3′,5′-cyclic monophosphate sodium salt N/A N/D N/D N/D N/D Cytidine 5′-monophosphate (CMP) active 5.7 0.3066 31.7 1.425 2′-Deoxyinosine 5′-monophosphate sodium salt unknown N/D N/D N/D N/D 2′-Deoxycytidine 5′-monophosphate N/A N/D N/D N/D N/D 2′-Deoxyguanosine 5′-monophosphate sodium salt hydrate active 0.24 0.5926 61.2 0.060 Guanosine 3′,5′-cyclic monophosphate sodium salt N/A N/D N/D N/D N/D Guanosine 5′-monophosphomorpholidate 4-morpholine-N,N′- active 1.3 0.5121 52.9 0.325 dicyclohexylcarboxamidine salt Inosine 3′:5′-cyclic monophosphate sodium salt N/A N/D N/D N/D N/D N6-Methyladenosine 5′-monophosphate sodium salt active 0.08 0.9907 102.3 0.020 D-Ribose 5-phosphate disodium salt hydrate unknown N/D N/D N/D N/D Sucrose 6′-monophosphate dipotassium salt unknown N/D N/D N/D N/D Thymidine 5′-monophosphate disodium salt hydrate (TMP) N/A N/D N/D N/D N/D Thiamine monophosphate chloride dihydrate unknown N/D N/D N/D N/D Uridine 5′-monophosphomorpholidate 4-morpholine-N,N′-dicyclohexylcarboxamidine active >3 1.0150 104.8 2.000 salt Uridine 5′-monophosphate disodium salt (UMP) active >30 0.2316 23.9 3.000 Abacavir 5′-Phosphate unknown N/D N/D N/D N/D Acyclovir Monophosphate N/A N/D N/D N/D N/D Acycloguanosine Monophosphate Ribavirin 5′-Monophosphate, Dilithium Salt active 0.7 1.5294 158.0 0.175 Tenofovir N/A N/D N/D N/D N/D 5′-Tosyl Adenosine unknown N/D N/D N/D N/D Xanthosine 5′-Monophosphate Disodium Salt (XMP) active 0.1 1.6223 167.6 0.025 Adenosine,5′-(dihydrogen phosphorothioate) active 0.011 1.2435 128.4 0.003 Adenosine-5′-O-monophosphorothioate (5′-AMPS) Cytidine-5′-O-monophosphorothioate (5′-CMPS) N/A N/D N/D N/D N/D Cytidine-3′-O-monophosphate (3′-CMP) N/A N/D N/D N/D N/D 7-Deazaadenosine-5′-O-monophosphate (7-CH-5′-AMP/5′-TuMP) active 0.75 0.4979 51.4 0.188 2′-Deoxyguanosine-5′-O-monophosphorothioate (5′-dGMPS) active 0.02 0.8446 87.2 0.005 2′-Deoxyuridine-5′-O-monophosphosphorothioate (5′-dUMPS) active >30 0.4200 43.4 5.000 2′-,3′-O-(N′-Methylanthraniloyl)guanosine-5′-O-monophosphate (MANT-5′-GMP) active 0.0056 0.7190 74.3 0.0014 Fluorescent analogue of 5′-GMP 6-Thioguanosine-5′-O-monophosphate (6-T-5′-GMP) active 0.0009 1.3614 140.6 0.0002 6-[(4-Nitrobenzyl)thio]-9-b-D-ribofuranosylpurine-5′-monophosphate disodium salt N/A N/D N/D N/D N/D Uridine 3′-monophosphate disodium salt N/A N/D N/D N/D N/D Adenosine 5′-monophosphomorpholidate 4-morpholine-N,N′- unknown N/D N/D N/D N/D dicyclohexylcarboxamidine salt Adenylosuccinic acid N/A N/D N/D N/D N/D 2′-Deoxyuridine 5′-monophosphate disodium salt N/A N/D N/D N/D N/D 2′-Deoxyadenosine 5′-monophosphate active 0.14 1.1503 118.8 0.035 Nicotinic acid mononucleotide unknown N/D N/D N/D N/D Orotidine 5′-monophosphate trisodium salt unknown N/D N/D N/D N/D Adefovir unknown N/D N/D N/D N/D Lamivudine 5′-monophosphate sodium salt N/A N/D N/D N/D N/D Guanosine monophosphate (GMP) - disodium salt active 0.03 1.2786 132.1 0.008 N-Lactoyl-5′-GMP active 0.1 1.3600 140.5 0.025 N-Acetyl-5′-GMP active 0.1 1.3351 137.9 0.025 N-gluconyl ethanolamine phosphate unknown N/D N/D N/D N/D Inosine 5′-monophosphate disodium salt (IMP) active 0.07 0.8681 89.7 0.018 Triciribine phosphate N/A N/D N/D N/D N/D Tricyclic Nucleoside Monophosphate (TCN-P) Guanosine 5′-triphosphate sodium salt hydrate active 0.7 1.8075 145.5 0.175 8-Bromoadenosine 5′-monophosphate N/A N/D N/D N/D N/D Cytidine 5′-diphosphocholine sodium salt hydrate N/A N/D N/D N/D N/D Guanosine 5′-diphosphate sodium salt active 0.2 1.7550 141.2 0.050 MRS 2179 ammonium salt hydrate active 0.05 1.8150 146.1 0.013 beta-Nicotinamide adenine dinucleotide hydrate unknown N/D N/D N/D N/D Thymidine 5′-monophosphate disodium salt hydrate unknown N/D N/D N/D N/D 6-Thioinosine Phosphate active 0.02 1.3875 111.7 0.005 ((2S,3R,4S)-5-(6-(benzylamino)-9H-purin-9-yl)-3-dihydroxytetrahydrofuran-2- unknown N/D N/D N/D N/D yl)methyl dihydrogen phosphate 2′-Deoxy-1,N6-ethenoadenosine-5′-O-monophosphate active 0.2 0.87 70.0 0.05 2′-Deoxycytidine-3′,5′-O-bisphosphate N/A N/D N/D N/D N/D 2-Amino-6-chloropurineriboside-5′-O-monophosphate active 0.0005 1.8700 150.5 0.00013 2′-Amino-2′-deoxyadenosine-5′-O-diphosphate unknown N/D N/D N/D N/D 3′-Amino-3′-deoxyadenosine-5′-O-diphosphate unknown N/D N/D N/D N/D N6-Benzyladenosine-5′-O-diphosphate_sodiumsalt unknown N/D N/D N/D N/D 8-Bromoguanosine-5′-O-monophosphate active CNBD 0.375 30.2 1 8-Chloroadenosine-5′-O-monophosphate,sodiumsalt active 0.8 0.79 63.6 0.2 6-Chloropurineriboside-5′-O-monophosphate,sodiumsalt active 0.002 1.5325 123.3 0.0005 2-Chloroadenosine-5′-O-monophosphate,sodiumsalt unknown N/D N/D N/D N/D N6-Cyclopentyladenosine-5′-O-diphosphate,sodiumsalt N/A N/D N/D N/D N/D 6-cPe-ADP 2′-Deoxyadenosine-5′-O-monophosphorothioate,sodiumsalt active 0.06 1.0375 83.5 0.015 5′-dAMPS 2′-Deoxyguanosine-5′-O-monophosphorothioate,sodiumsalt active 0.01 1.0775 86.7 0.0025 2′-Deoxyinosine-5′-O-monophosphorothioate,sodiumsalt active 0.1 1.0375 83.5 0.025 2′-Deoxy-3′-O-(N′-methylanthraniloyl)adenosine-5′-O-monophosphate active 0.01 0.3975 32.0 0.0025 2′-Deoxy-3′-O-(N′-methylanthraniloyl)guanosine-5′-O-monophosphate active 0.01 0.295 23.7 0.0025 2′-Deoxy-3′-O-(N′-methylanthraniloyl)guanosine-5′-O-diphosphate N/A N/D N/D N/D N/D MANT-dGDP 2′-Deoxyguanosine-3′,5′-O-bisphosphate active 0.02 1.8925 152.3 0.005 1,N6-Ethenoadenosine-5′-O-diphosphate,sodiumsalt unknown N/D N/D N/D N/D Guanosine-5′-O-(1-thiodiphosphate),sodiumsalt,Sp-isomer N/A N/D N/D N/D N/D Guanosine-5′-monophosphorothioate sodium salt active 0.01 1.29 103.8 0.0025 5′-GMPS Guanosine-5′-O-(2-thiodiphosphate) active 0.01 0.5875 47.3 0.0025 Guanosine-5′-O-(a,ß-methylene)diphosphate N/A N/D N/D N/D N/D 2-Methylthioadenosine-5′-O-diphosphate,sodiumsalt unknown N/D N/D N/D N/D N6-(1-Methylbutyl)-ADP,sodiumsalt N/A N/D N/D N/D N/D 2′-/3′-O-(N′-Methyl-anthraniloyl)adenosine-5′-O-monophosphate sodium salt active 0.01 1.915 154.1 0.0025 MANT-AMP N6-(2-Phenylethyl)adenosine-5′-O-diphosphate sodium salt N/A N/D N/D N/D N/D 6-PhEt-ADP N6-Phenyladenosine-5′-O-diphosphate,sodiumsalt N/A N/D N/D N/D N/D 2-Methylthioadenosine-5′-O-monophosphate sodium salt unknown N/D N/D N/D N/D 2-MeS-AMP Inosine 5′-diphosphate (IDP) sodium salt active 0.65 0.3 33.3 0.1625 2′-/3′-O-(2-Aminoethylcarbamoyl)adenosine-5′-O-monophosphate; 2′-/3′-AEC-5′-AMP active 0.09 2.2 213.9 0.0225 2′-/3′-O-(2-Aminoethylcarbamoyl) active 0.14 1.1 109.5 0.035 guanosine-5′-O-monophosphate; 2′-/3′-AEC-5′-GMP Inosine Triphosphate (ITP) trisodium salt active 0.08 0.3 25.1 0.02 N6-Benzoyladenosine-5′-O- active >1 1.1 105.6 0.3 monophosphate; 6-Bnz-5′-AMP 7-Deaza-2′-deoxyadenosine-5′-O- N/A N/D N/D N/D N/D monophosphate (7-CH-5′-dAMP/ 5′-dTuMP) 2′-Deoxyadenosine-3′,5′-O-bisphosphate active 0.02 0.8 76.4 0.005 N6-(6-Aminohexyl)adenosine 2′,5′-diphosphate N/A N/D N/D N/D N/D Adenosine 5′-O-thiomonophosphate dilithium salt active 0.06 1.4 139.4 0.015 Riboflavin Phosphate Sodium Salt unknown N/D N/D N/D N/D Cytidine sulphate N/A N/D N/D N/D N/D 2′-Deoxyuridine-5′-O-monophosphorothioate (5′-dUMPS) N/A N/D N/D N/D N/D 2′-Deoxycytidine-5′-O-monophosphorothioate (5′-dCMPS) unknown N/D N/D N/D N/D Uridine-5′-O-monophosphorothioate (5′-UMPS) N/A N/D N/D N/D N/D N/D = Not determined N/T = Not tested N/A = Not active CNBD = Could not be determined

TABLE 16 Positive and negative controls. Amino acid EC50's were measured in the presence of 0.2 mM IMP. Type of control Compound Name EC₅₀ (mM) Positive L-Alanine 13.6 ± 4.3 mM L-Serine 31.5 ± 2.3 mM L-Cysteine 39.5 ± 1.9 mM L-Phenylalanine 1.9 ± 0.1 mM L-Tryptophan 0.9 ± 0.1 mM L-Histidine 10.2 ± 1.7 mM L-Methionine 25.5 ± 3.7 mM L-Tyrosine 10.3 ± 0.9 mM Negative L-Proline — MSG — Quinine HCL — D-Glucose —

Example 3—Identification of T1R1 Nucleotide and Amino Acid Interacting Domains

The present example describes the in silico identification of amino acids within T1R1 that interact with nucleotides and amino acids that bind to T1R1.

Methods:

The cat T1R1 is a Group C G protein-coupled receptor (GPCR) that complexes with T1R3 to form an umami taste receptor heterodimer. A model of the cat T1R1 Venus Flytrap domain (VFT domain) was built using the crystal structure of metabotropic glutamate receptor 1EWT, another Group C GPCR (Kunishima et al., Nature 407: 971-977 (2000)), which is available from the Protein Data Bank (Berman et al., Nucleic Acids Research, 28: 235-242 (2000)). Crystal structures of VFT's of Group C GPCR's, including metabotropic glutamate receptors mGluR1, mGluR3, mGluR5, and mGluR7, show remarkably similar modes of ligand binding to the active site cleft of the VFT's. These ligand binding modes were used to manually align the cat T1R1 VFT sequence to the sequence profile of metabotropic glutamate receptors. Subsequently, this alignment was used to perform homology modeling using the Modeller software package (Eswar et al., Curr Protoc Bioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30 (2006)).

Modeling Amino Acids into the Active Site of T1R1:

Alanine (L-alanine) was initially positioned in the active site of the cat T1R1 VFT model following the placement of the zwitterionic backbone of glutamate in mGluR crystal structures. The resulting complex was refined using molecular dynamics and energy minimization. The binding energy was estimated by calculating the difference between the calculated energy of the complex and the calculated energy for the isolated ligand and apo protein using a continuum model for water. Other amino acids were built onto the bound alanine scaffold using Discovery Studio (Dassault Systemes, BIOVIA Corp., San Diego, Calif., USA), and refined using molecular dynamics and energy minimization (Brooks et al., J Comput Chem. 30(10):545-614 (2009)). Final models were selected for the amino acids as those whose calculated binding energies were comparable to that of alanine, and that also retained conserved interactions to the hinge observed in mGluR crystal structures.

Modeling Nucleotides into the Active Site of T1R1:

IMP and GMP were initially positioned into the cat T1R1 VFT active site following previously published modeling of IMP into human T1R1, such as described by Zhang et. al. (Zhang et al., Proc Natl Acad Sci USA. 105(52):20930-4 (2008)). Nucleotide torsional bonds were varied and resulting models refined using molecular dynamics and energy minimization. Final models were selected as those whose binding energies were comparable to that of GMP, and that also exhibited nucleotide interactions to conserved residues established as important for IMP binding to human T1R1 by site-directed mutagenesis (Zhang et al., Proc Natl Acad Sci USA. 105(52):20930-4 (2008)). Other nucleotide and nucleotide analogs were initially overlapped with the IMP and GMP models and then subjected to the same refinement, evaluation, and selection procedure described for IMP and GMP.

Results:

The cat T1R1 VFT domain consists of two lobes as shown in FIG. 14. The upper lobe and the lower lobe are connected by three strands of protein called the hinge. In FIG. 14, the upper lobe is to the top in the figure; the lower lobe is to the bottom in the figure. The hinge is to the left. The flytrap domain transitions from an open to a closed conformation on agonist binding. Active amino acids and nucleotides bind to the VFT domain between the two lobes (see FIG. 14). Amino acids bind to a region near the hinge (see FIG. 14: alanine is shown in CPK space filling rendering to the left in the figure). Nucleotides bind to a region more distal to the hinge but still located between the lobes of the flytrap (see FIG. 14: IMP is shown in CPK space filling rendering to the right in the figure).

Amino Acid Binding:

Amino acid ligands bind to the hinge region of the VFT as shown in FIG. 14, and are well-coordinated by interactions near the hinge. FIG. 15 shows an example binding mode for L-alanine illustrating possible hydrogen bonds, salt-bridge interactions, and Pi-cation interactions to Thr149, Ser172, Tyr220, Thr148, Glu170, and Asp302. These interactions are shown as dotted lines.

Thr149, Ser172, and Tyr220 residues were established as important to L-glutamate binding in the human umami receptor by site-directed mutagenesis (Zhang et al., Proc Natl Acad Sci USA. 105(52):20930-4 (2008)). Mouse Asn149, which corresponds to Thr148 in cat, was shown to be important to amino acid binding in mouse (Toda et. al., J. Biol. Chem 288:36863-36877 (2013)). Glu170 and Asp302 are present in cat and mouse T1R1, but not in human. In humans, the amino acids at these positions are alanine. Human T1R1/T1R3 is highly selective for L-Glutamate and L-Aspartic Acid. By contrast, cat and mouse T1R1/T1R3 respond to a broad range of amino acids. In the model described by the present example, Glu170 and Asp302 help coordinate the zwitterionic nitrogen of amino acid ligands that fit the active site of T1R1, while simultaneously establishing an electrostatic environment that is inhospitable to binding L-Glutamate and L-Aspartic Acid. Using site directed mutagenesis Toda et. al. demonstrated that Glu170 and Asp302 are responsible for the observed difference in amino-acid ligand preferences between cat and human (Toda et. al., J. Biol. Chem 288:36863-36877 (2013)).

Nucleotide Binding:

Nucleotides bind to a position that is more distal to the hinge than do amino acids, as illustrated in FIG. 14. FIG. 16 shows an example binding mode for GMP. Possible hydrogen bond and salt bridge interactions to the phosphate of GMP and T1R1 His47, His71, Arg277, and Asn69; the sugar of GMP and T1R1 Asn302 and Ser306; and the GMP base and T1R1 Ser384, His308, and Ala380. These interactions are shown as dotted lines.

His308 is shown coordinating the base of GMP but may also swing to coordinate GMP's phosphate. Additional hydrophobic interactions that add to the interaction energy of the ligand to the VFT are also present, and the flexibility of the different binding sites can vary (data not shown). Different nucleotides may exhibit different interactions with T1R1, but may overlap with the interactions described herein for GMP. Structure-activity relationships (SAR) and the T1R1 model suggest that the presence of a negatively charged group in the phosphate binding region of T1R1 is important to the binding of a nucleotide to T1R1. SAR and the model suggest that the presence of a nucleotide base, an extended base, a substituted base, or other bioisosteric replacement for the nucleotide base that can form interactions in the nucleotide base binding region of T1R1 is also important for binding (for examples of bases see Limbach et. al., Nucleic Acids Research 22(12): 2183-2196 (1994)). Similarly SAR and modeling suggest that the interaction between a nucleotide sugar (or sugar replacement molecule) is important for successful binding of a nucleotide to T1R1. The sugar can orient an appropriate negatively charged group to also help establish interactions between T1R1 and the phosphate region and nucleotide base region of the nucleotide.

Although different nucleotides may exhibit different interactions with T1R1, such interactions would likely conform in large part to this set of possible interactions.

Bridging Interactions Between Amino Acid and Nucleotide Binding:

Asp302 is a residue present in the VFT of T1R1 in multiple species, including cat and mouse. However, in humans the amino acid at this position is alanine. Asp302 has a flexible side chain that may orient to coordinate the zwitterionic nitrogen or side-chain of a bound amino acid ligand (FIG. 15) or may alternatively orient to coordinate the sugar of a bound nucleotide (FIG. 16). Furthermore, Asp302 may also be oriented to simultaneously coordinate the zwitterionic backbone nitrogen of a bound amino acid, and the sugar of a nucleotide (FIG. 17). This bridging interaction may enhance the synergistic effect between a bound amino acid and a bound nucleotide. Because the amino acid at this position in humans is alanine, such a bridging interaction is not possible in humans. An alternative conformation of the nucleotide base may establish further bridging interactions between Glu170 and select nucleotide bases (data not shown).

Example 4—In Silico Identification of Transmembrane Compounds

The present example describes the computational modeling of the T1R1/T1R3 receptor to identify putative transmembrane compounds.

Computational approaches were used to analyze the three-dimensional structure of T1R1 to identify transmembrane regions that can be exploited to selectively activate the T1R1/T1R3 receptor. Although the crystal structure of cat T1R1 has not been determined, a structural model of the transmembrane region of T1R1 was generated based on the crystal structure of the human GPCR Metabotropic Glutamate Receptor 1 (mGluR1) (Wu et al., 2014 Science Vol. 344, p. 58-64) and of the human GPCR Metabotropic Glutamate Receptor 5 (mGluR5) (Dore et al., Nature. 2014 Jul. 31; 511(7511):557-62. Epub 2014 Jul. 6). “In silico” modeling was then used to identify small chemical compounds that could potentially interact with the transmembrane domain of the T1R1 monomer of the T1R1/T1R3 receptor.

FIG. 28 shows the interaction of N-benzyl-L-phenylalanine methyl ester with T1R1. Asparagine (Asn) 735 of the T1R1 transmembrane domain can be seen interacting with the ester of the ligand. A cluster of aryl residues is positioned in the active site that can coordinate with the ligand by forming ring-stacking interactions to the phenyl group of the ligand to the right in the figure. FIG. 35 shows the modeling of 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione within the transmembrane region of T1R1. Phe642 of Helix 6 and Phe776 of Helix 2 interact with the phenyl ring of 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione and Asn792 of Helix 6 interacts with the carbonyl group by a hydrogen bond (FIG. 35). The modeling of 1H-imidazo[4,5-c]pyridin-2(3H)-one within the transmembrane region of T1R1 shows the hydrogen bond between Asn735 of the transmembrane region of T1R1 and 1H-imidazo[4,5-c]pyridin-2(3H)-one (FIG. 36).

Example 5—Activation of T1R1/T1R3 Receptor by Transmembrane Compounds

The present example describes the activation of the T1R1/T1R3 receptor by transmembrane compounds in vitro.

Based on the in silico modeling described in Example 4, putative T1R1/T1R3 transmembrane compounds were identified and selected for further testing in vitro. In vitro functional characterization of the selected compounds was used to evaluate the effectiveness of the putative transmembrane compounds in activating the T1R1/T1R3 receptor alone or in combination with one or more nucleotides and/or one or more amino acids.

Methods:

HEK293 cells that stably express T1R3 and inducibly express T1R1 were exposed to transmembrane compounds alone or in combination with one or more amino acids and/or one or more nucleotides to activate the umami receptor. Activation of the T1R1/T1R3 receptor was detected by a change in intracellular calcium levels using a calcium sensitive fluorescent dye. Cells that express T1R3 but not T1R1 were used as a control. A FLIPR® Tetra or a FlexStation® 3 was used for data capture.

For each transmembrane compound, dose response curves were generated and the following properties were determined: EC₅₀ of the transmembrane compound alone; EC₅₀ of the transmembrane compound with 20 mM alanine; EC₅₀ of the transmembrane compound with 0.2 mM IMP; and EC₅₀ of the transmembrane compound with 20 mM alanine and 0.2 mM IMP. The term half maximal effective concentration (EC₅o) refers to the concentration of a compound which induces a response halfway between the baseline and the maximum after a specified exposure time. In each experiment, serial dilutions of up to 10 mM of the transmembrane compound were added to the T1R1/T1R3-expressing cells.

Results:

Treatment of HEK293 cells expressing T1R1/T1R3 receptors with 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione alone (e.g., in buffer) or in combination with 20 mM alanine resulted in the activation of the T1R1/T1R3 receptor, as indicated by the change in intracellular calcium levels (ΔF/F₀), and resulted in an observed EC₅₀ value greater than 1 mM. In contrast, 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione, in the presence of 0.2 mM IMP or in the presence of 20 mM alanine and 0.2 mM IMP, resulted in a decrease in the EC₅₀ value to 0.32±0.05 mM and 0.33±0.04, respectively (FIG. 37 and Table 17). These results indicate that IMP is a positive allosteric modulator of the transmembrane compound 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5-trione, i.e. that the compounds are synergistic in effect on T1R1/T1R3.

The compound, N-(benzo[d][1,3]dioxol-5-yl)-2-propylpentanamide, was observed to function as an agonist of T1R1/T1R3 alone (FIG. 39 and Table 17). In the presence of IMP or IMP and alanine, the EC₅₀ of N-(benzo[d][1,3]dioxol-5-yl)-2-propylpentanamide decreased and the ΔF/F₀ extended significantly higher (FIG. 39 and Table 17). These results suggest that alanine and IMP act synergistically with N-(benzo[d][1,3]dioxol-5-yl)-2-propylpentanamide to activate T1R1/T1R3. Similar results were observed for the transmembrane compound N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide (FIG. 40 and Table 17). IMP functioned as a positive allosteric modulator of N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide.

The putative transmembrane compound, N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide, failed to activate T1R1/T1R3 alone; however, in the presence of IMP or IMP and alanine, N-(2-amino-2-oxo-1-phenylethyl)-3-chloro-4,5-dimethoxybenzamide activated T1R1/T1R3 as indicated by the increase in ΔF/F₀ at lower concentrations and the reduction in EC₅₀ (FIG. 41 and Table 17). Similar results were observed with 2-((5-(4-(methylthio)phenyl)-2H-tetrazol-2-yl)methyl)pyridine (FIG. 43 and Table 17).

The compound, (E)-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide, functions as a transmembrane compound of T1R1/T1R3 with an EC₅₀ of 0.45±0.01. In the presence of IMP or IMP and alanine, the activity of (E)-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide was greatly enhanced resulting in an EC₅₀ of 0.15±0.02 and 0.08±0.01, respectively, indicating that IMP and alanine function as allosteric modulators of (E)-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (FIG. 42 and Table 17).

The transmembrane compound, N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide, was further analyzed. FIG. 44 shows the dose response curves of N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide in the presence of GMP and phenylalanine; GMP, phenylalanine and alanine; GMP, phenylalanine and IMP; or GMP, phenylalanine, alanine and IMP. The presence of alanine and IMP significantly decreased the EC₅₀ value of N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide compared to the EC₅o value N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide in the presence of phenylalanine and GMP or in the presence of IMP alone (Table 19). As indicated above, IMP functions as an allosteric modulator of the agonist activity of N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide; however, these results indicate that combination of IMP and alanine resulted in a lower EC₅₀ value and exhibited a higher shift in ΔF/F₀ than IMP alone, indicating that this combination displayed a synergistic effect with N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide in the activation of T1R1/T1R3 (FIG. 44 and Table 19). In addition, the ΔF/F₀ is approximately 10×higher in the presence of the ternary mixture of N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide, phenylalanine and GMP, compared to combination of phenylalanine and GMP in the absence of the agonist (FIG. 45).

FIG. 46 shows the dose response curves for the positive and negative controls used, and the results for active compounds are summarized in Table 17. Table 18 shows the results for all compounds tested. For the positive and negative controls, dose response curves for amino acids were determined in the presence of 0.2 mM IMP. Dose response curves for nucleotides were determined in the presence of 20 mM alanine.

Without being bound to a particular theory, these results show that nucleotides alone, e.g., IMP, or combinations of nucleotides and amino acids, e.g., IMP and alanine, function as positive modulators of the disclosed transmembrane compounds, thereby resulting in the reduction in the amount of the agonist necessary to sufficiently activate the T1R1/T1R3 receptor.

TABLE 17 Transmembrane compounds that modulate T1R1/T1R3 activity + Alanine (20 mM) + + Alanine (20 nM) + IMP (0.2 mM) IMP + buff-er Max Max (0.2 (alone) Max resp Max resp mM) Compound EC₅₀ EC₅₀ resp % of Thres EC₅₀ resp % of Thres EC₅₀ Name Chemical Structure (mM) (mM) ΔF/F IMP (mM) (mM) ΔF/F ALa (nM) (mM) 5-bromo- N- (pentan-3- yl)furan-2- carbox- amide

>3  >3  1.575  126.8 1  >1  1.8425 149.6 0.3   >1  3,4- dimethyl- N-(pentan- 3-yl) benzamide; N-(1-ethyl- propyl)-3,

>3  >3  1.2150  97.8 3  2.1 1.5475 125.7 0.525  1.7 4-dimethyl- benzamide 4- methoxy- 3-methyl- N-(1-phenyl- ethyl) benzamide

>3  >3  0.51   41.0 3  1.4 1.1775  95.6 0.35  1.2 N-(2,3- dimethyl- cyclohexyl) benzo[d] [1,3] dioxole-5- carbox- amide

2  >3  0.225   18.1 3  1.1 1.175   95.4 0.275  0.6 ethyl 2- (benzo[d] [1,3] dioxole-5- carbox- amido)- 3-methyl- butanoate;

>10   >10   0.3715  25.6 10  >3  0.65   52.8 1    >3  ethyl N- (1,3- benzo- dioxol- 5-yl- carbonyl) valinate 4-methoxy- 3-methyl- N- (pentan-2- yl) benzamide

0.4 0.2 1.5650 126.0  0.050 0.1 1.5875 128.9 0.025   0.03 N-(pentan- 3-yl)-2,3- dihydro- benzo [b][1,4] dioxine-6- carboxamide

>1  >1  0.3125  25.2 0.3 0.3 0.8725  70.9 0.075  0.2 3,4- dimethoxy- N-(pentan- 3-yl) benzamide

>10   >10   0.545   43.9 0.3 2  0.9975  81.0 0.5   >3  (E)-methyl 3-methyl- 2-(3- (thiophen- 2-yl)acryl- amido)

CNBD CNBD 0.1175  9.5 10  >10   0.7375  59.9 1    >10   butanoate (E)-3-(3,4- dimethoxy- phenyl)-N- (pentan- 2-yl) acrylamide;

>10   >10   0.4375  35.2 1  2.2 1.07   86.9 0.55  1.9 (2E)-3-(3,4- dimethoxy- phenyl)- N-(pentan- 2-yl)prop- 2-enamide (E)-3-(4- methoxy- phenyl)-N- (2-methyl- cyclohexyl) acrylamide

>10   >10   0.785   63.2 3  >3  1.7725 144.0 0.3   >3  N1- phenethyl- N2-(pyridin- 3-ylmethyl) oxalamide;

>1  >1  0.3750  30.2 1  >0.3  0.7625  61.9 0.1   0.2 N-(2-phenyl- ethyl)-N′- (pyridin-3- ylmethyl) ethane- diamide N-(heptan- 4-yl) benzo[d] [1,3] dioxole-5- carboxamide

0.7 1.1 1.7725 142.7  0.275  0.09 1.4725 119.6 0.225  0.1 1-(2-bromo- phenyl)- 3-((1R,2S)- 2-hydroxy- 2,3-dihydro- 1H-inden- 1-yl)urea

>3  >3  0.3    33.0 3  >3  0.378   44.9 3    >3  N- (benzo[d] [1,3] dioxol-5- yl)-2- propyl- pent- anamide

 0.35 CNBD CNBD CNBD CNBD  0.27 0.908  108.0 0.0675  0.09 1-benzyl-3- (2-oxo-2- phenyl- ethyl)imi- dazolidine- 2,4,5-trione

>1  >1  1.3   127.6 1   0.32 1.140  135.7 0.08   0.33 N-(2- amino- 2-oxo-1- phenyl- ethyl)- 3-chloro- 4,5- dimethoxy- benzamide

CNBD CNBD CNBD CNBD CNBD  0.68 0.415   49.4 0.17   0.75 (E)-3-(4- methoxy- phenyl)-N- (pentan-3- yl) acrylamide

 0.45  0.59 0.7    64.1  0.148  0.15 1.443  171.7 0.0375  0.08 2-((5-(4- (methylthio) phenyl)- 2H- tetrazol- 2-yl) methyl) pyridine

CNBD CNBD CNBD CNBD CNBD  0.63 0.678   80.7 0.1575  0.27 N- (heptan-4- yl)benzo[d] [1,3] dioxole-5- carboxamide

 0.26  0.42 1.2   118.2  0.105 0.1 1.393  165.8 0.025   0.03 CNBD: Could not be determined

TABLE 18 Activation of T1R1/T1R3 by transmembrane compounds +Alanine (20 mM) + +buffer +Alanine (20 mM) +IMP (0.2 mM) IMP (alone) Max Max Max Max (0.2 mM) EC₅₀ EC₅₀ resp resp % Thres EC₅₀ resp resp % Thres EC₅₀ Compound Name Activity (mM) (mM) ΔF/F of IMP (mM) (mM) ΔF/F of Ala (mM) (mM) 5-bromo-N-(pentan-3-yl)furan-2- active >3 >3 1.575 126.8 1 >1 1.8425 149.6 0.3 >1 carboxamide 3,4-dimethyl-N-(pentan-3-yl)benzamide; active >3 >3 1.2150 97.8 3 2.1 1.5475 125.7 0.525 1.7 N-(1-ethylpropyl)-3,4-dimethylbenzamide N-(2,4-dimethylpentan-3-yl)-4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methoxybenzamide 4-ethoxy-N-(pentan-3-yl)benzamide N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 4-methoxy-3-methyl-N-(1- active >3 >3 0.51 41.0 3 1.4 1.1775 95.6 0.35 1.2 phenylethyl)benzamide N-(2,3- active 2 >3 0.225 18.1 3 1.1 1.175 95.4 0.275 0.6 dimethylcyclohexyl)benzo[d][1,3]dioxole-5- carboxamide methyl 2-(benzo[d][1,3]dioxole-5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D carboxamido)-4-methylpentanoate; methyl N-(1,3-benzodioxol-5- ylcarbonyl)leucinate ethyl 2-(benzo[d][1,3]dioxole-5- active >10 >10 0.3175 25.6 10 >3 0.65 52.8 1 >3 carboxamido)-3-methylbutanoate; ethyl N-(1,3-benzodioxol-5- ylcarbonyl)valinate 4-methoxy-3-methyl-N-(pentan-2- active 0.4 0.2 1.5650 126.0 0.050 0.1 1.5875 128.9 0.025 0.03 yl)benzamide N-(pentan-3-yl)-2,3- active >1 >1 0.3125 25.2 0.3 0.3 0.8725 70.9 0.075 0.2 dihydrobenzo[b][1,4]dioxine-6-carboxamide 3,4-dimethoxy-N-(pentan-3-yl)benzamide active >10 >10 0.545 43.9 0.3 2 0.9975 81.0 0.5 >3 (E)-methyl 3-methyl-2-(3-(thiophen-2- active CNBD CNBD 0.1175 9.5 10 >10 0.7375 59.9 1 >10 yl)acrylamido)butanoate (E)-3-(3,4-dimethoxyphenyl)-N-(pentan-2- active >10 >10 0.4375 35.2 1 2.2 1.07 86.9 0.55 1.9 yl)acrylamide; (2E)-3-(3,4-dimethoxyphenyl)-N-(pentan-2- yl)prop-2-enamide (E)-3-(4-methoxyphenyl)-N-(2- active >10 >10 0.785 63.2 3 >3 1.7725 144.0 0.3 >3 methylcyclohexyl)acrylamide N-(2,3-dihydro-1H-inden-1- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D yl)benzo[d][1,3]dioxole-5-carboxamide N1-phenethyl-N2-(pyridin-3- active >1 >1 0.3750 30.2 1 >0.3 0.7625 61.9 0.1 0.2 ylmethyl)oxalamide; N-(2-phenylethyl)-N′-(pyridin-3- ylmethyl)ethanediamide N-benzyl-N′-(2-phenylethyl)ethanediamide N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 1-(4-chlorophenyl)-3-[1-(4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methylphenyl)propyl]urea; 1-(4-chlorophenyl)-3-(1-(p-tolyl)propyl)urea 1-(2-chlorophenyl)-3-cyclohexylurea N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (E)-N-(2,4-dimethylpentan-3-yl)-3-(4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D fluorophenyl)acrylamide 5-bromo-N-cycloheptyl-2-furamide; N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 5-bromo-N-cycloheptylfuran-2-carboxamide N-cyclohexyl-1,3-benzodioxole-5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D carboxamide N-(2- un N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (dimethylamino)ethyl)benzo[d][1,3]dioxole- known 5-carboxamide N-(2-(methylamino)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D oxoethyl)benzo[d][1,3]dioxole-5- carboxamide N-(heptan-4-yl)benzo[d][1,3]dioxole-5- active 0.7 1.1 1.7725 142.7 0.275 0.09 1.473 119.6 0.225 0.1 carboxamide 1-(2-bromophenyl)-3-((1R,2S)-2-hydroxy- active >3 >3 0.3 33.0 3 >3 0.378 44.9 3 >3 2,3-dihydro- 1H-inden-1-yl)urea 3-(7-bromo-2,3-dihydrobenzo[b][1,4]dioxin- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 6-yl)- 1-(1-(4-chlorophenyl)ethyl)-1-methylurea 1-(2,4-dichlorophenyl)-3-(4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methoxybenzyl)urea (E)-N-cyclohexyl-3-(4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D propoxyphenyl) acrylamide (E)-N-cyclohexyl-3-(pyridin-4-yl) N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D acrylamide N-(benzo[d][1,3]dioxol-5-yl)-2- active 0.35 CNBD CNBD CNBD CNBD 0.27 0.908 108.0 0.0675 0.09 propylpentanamide N1-(4-methoxybenzyl)-N2- un- N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenethyloxalamide known 1-benzyl-3-(2-oxo-2- active >1 >1 1.3 127.6 1 0.32 1.140 135.7 0.08 0.33 phenylethyl)imidazolidine-2,4,5-trione N1-(3,4-dimethoxyphenethyl)-N2-(pyridin- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 3-ylmethyl)oxalamide N-(2-amino-2-oxo-1-phenylethyl)-3-chloro- active CNBD CNBD CNBD CNBD CNBD 0.68 0.415 49.4 0.17 0.75 4,5-dimethoxybenzamide (E)-3-(4-methoxyphenyl)-N-(pentan-3- active 0.45 0.59 0.7 64.1 0.148 0.15 1.443 171.7 0.0375 0.08 yl)acrylamide (E)-3-(3-chloro-4,5-dimethoxyphenyl)-N- N/A cyclohexylacrylamide 1-(4-chlorobenzyl)-4-phenethylpyrazine- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2,3(1H,4H)-dione 1-((3-methoxybenzyl)thio)-4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenylphthalazine 3-(benzo[d][1,3]dioxol-5-yl)-6-((4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D bromobenzyl)thio)pyridazine 5-(3,5-dichlorophenyl)-N-((3-methylpyridin- un N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2-yl)methyl)-1,3,4-thiadiazol-2-amine known 2-((5-(4-(methylthio)phenyl)-2H-tetrazol-2- active CNBD CNBD CNBD CNBD CNBD 0.63 0.678 80.7 0.1575 0.27 yl)methyl)pyridine 5-((2-chlorobenzyl)thio)-3-(4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methoxyphenyl)-1H-1,2,4-triazole 2-(((5-(p-tolyl)-4H-1,2,4-triazol-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D yl)thio)methyl)pyridine N-(heptan-4-yl)benzo[d][1,3]dioxole-5- active 0.26 0.42 1.2 118.2 0.105 0.1 1.393 165.8 0.025 0.03 carboxamide N/D = Not determined N/T = Not tested N/A = Not active CNBD = Could not be determined

TABLE 19 Activation of T1R1/T1R3 by combinations of nucleotides, amino acids and transmembrane compounds EC₅₀ - with Ala EC₅₀ - alone EC₅₀ - with EC₅₀ - with (20 mM) and Compound Name (mM) Ala (20 mM) IMP (0.2 mM) IMP (0.2 mM) GMP 0.04 ± 0.01 0.05 ± 0.02 — — Phenylalanine — — 3.4 ± 0.7 — N-(heptan-4-yl)benzo[d][1,3]dioxole-5- 0.26 ± 0.09 0.42 ± 0.06 0.09 ± 0.04 0.03 ± 0.03 carboxamide GMP + Phenylalanine (1:30) 0.26 ± 0.04 0.29 ± 0.08 0.23 ± 0.04 — mM GMP + Phenylalanine (1:3) >0.3 0.11 ± 0.02 — — GMP + Phenylalanine (1:0.3) 0.05 ± 0.01 0.09 ± 0.01 — — GMP + Phenylalanine + N-(heptan-4- 0.011 ± 0.001 0.008 ± 0.001  0.006 ± 0.0004 0.005 ± 0.001 yl)benzo[d][1,3]dioxole-5-carboxamide (1:30:10)

TABLE 20 Positive and negative controls measured with 0.2 mM IMP. EC₅₀ (effect with Type of control Compound Name 0.2 mM IMP) Positive L-Alanine 13.6 ± 4.3 mM L-Serine 31.5 ± 2.3 mM L-Cysteine 39.5 ± 1.9 mM L-Phenylalanine 1.9 ± 0.1 mM L-Tryptophan 0.9 ± 0.1 mM L-Histidine 10.2 ± 1.7 mM L-Methionine 25.5 ± 3.7 mM L-Tyrosine 10.3 ± 0.9 mM IMP 0.08 ± 0.002 mM GMP 0.05 ± 0.004 mM Negative L-Proline — MSG — Quinine HCL — D-Glucose —

Example 6—Activation of T1R1/T1R3 Receptor by Transmembrane Compounds

The present example describes the activation of the T1R1/T1R3 receptor by transmembrane compounds in vitro.

Based on the in silico modeling described in Example 4, putative T1R1/T1R3 transmembrane compounds were identified and selected for further testing in vitro. In vitro functional characterization of the selected compounds was used to evaluate the effectiveness of the putative transmembrane compounds in activating the T1R1/T1R3 receptor alone or in combination with one or more nucleotides and/or one or more amino acids.

Methods: In vitro functional characterization of the selected compounds was conducted as described by Example 5.

Results: As shown in Table 21, treatment of HEK293 cells expressing T1R1/T1R3 receptors with N-benzyl-L-phenylalanine methyl ester HCl alone (e.g., in buffer) resulted in the activation of the T1R1/T1R3 receptor with an EC₅₀ of 0.03±0.002 mM. Combining the compound with 20 mM L-alanine increased the EC₅₀ to 0.05±0.001 mM. Combining the compound with 0.2 mM IMP lowered the EC₅₀ to 0.02±0.001 mM, and combining the compound with 20 mM L-alanine and 0.2 mM IMP lowered the EC₅₀ to 0.02±0.021 Mm.

TABLE 21 EC₅₀ of N-benzyl-L-phenylalanine methyl ester HCl in activating T1R1/T1R3.

                      T1R1/T1R3 EC₅₀ alone in buffer 0.03 ± 0.002 mM +20 mM L-alanine 0.05 ± 0.001 mM +0.2 mM IMP 0.02 ± 0.001 mM +20 mM L-alanine + 0.2 mM IMP 0.02 ± 0.002 mM

As shown in Table 22, treatment of HEK293 cells expressing T1R1/T1R3 receptors with N-(2-(1H-indol-3-yl)ethyl)nicotinamide alone (e.g., in buffer) resulted in the activation of the T1R1/T1R3 receptor with an EC₅₀ of 0.15±0.03 mM. Combining the compound with 20 mM L-alanine increased the EC₅₀ to greater than 0.1 mM. Combining the compound with 0.2 mM IMP lowered the EC₅₀ to 0.05±0.01 mM, and combining the compound with 20 mM L-alanine and 0.2 mM IMP lowered the EC₅₀ to 0.04±0.01 mM.

TABLE 22 EC₅₀ of N-(2-(1H-indol-3-yl)ethyl)nicotinamide in activating T1R1/T1R3.

                T1R1/T1R3 EC₅₀ alone in buffer 0.15 ± 0.03 mM +20 mM L-alanine >0.1 mM +0.2 mM IMP 0.05 ± 0.01 mM +20 mM L-alanine + 0.2 mM IMP 0.04 ± 0.01 mM

As shown in Table 23, treatment of HEK293 cells expressing T1R1/T1R3 receptors with 2-amino-N-phenethylbenzamide alone (e.g., in buffer) resulted in the activation of the T1R1/T1R3 receptor with an EC₅₀ of 0.42±0.01 mM. Combining the compound with 20 mM L-alanine increased the EC₅₀ to 0.48±0.01 mM. Combining the compound with 0.2 mM IMP lowered the EC₅o to 0.14±0.03 mM, and combining the compound with 20 mM L-alanine and 0.2 mM IMP lowered the EC₅₀ to 0.09±0.01 mM.

TABLE 23 EC₅₀ of 2-amino-N-phenethylbenzamide in activating T1R1/T1R3.

                  T1R1/T1R3 EC₅₀ alone in buffer 0.42 ± 0.01 mM +20 mM L-alanine 0.48 ± 0.01 mM +0.2 mM IMP 0.14 ± 0.03 mM +20 mM L-alanine + 0.2 mM IMP 0.09 ± 0.01 mM

Without being bound to a particular theory, these results show that nucleotides alone, e.g., IMP, or combinations of nucleotides and amino acids, e.g., IMP and alanine, function as positive modulators of the disclosed transmembrane compounds, thereby resulting in the reduction in the amount of the agonist necessary to sufficiently activate the T1R1/T1R3 receptor.

Example 7—Activation of T1R1/T1R3 Receptor by Transmembrane Compounds and Nucleotide Derivatives

The present example describes the activation of the T1R1/T1R3 receptor by transmembrane compounds and nucleotide derivatives in vitro.

Based on the in silico modeling described in Example 4, putative T1R1/T1R3 transmembrane compounds were identified and selected for further testing in vitro. In vitro functional characterization of the selected compounds was used to evaluate the effectiveness of the putative transmembrane compounds in activating the T1R1/T1R3 receptor alone or in combination with one or more nucleotides and/or one or more amino acids. Select nucleotide derivatives were also tested alone or in combination with one or more nucleotides and/or one or more amino acids.

Methods: In vitro functional characterization of the selected compounds was conducted as described by Examples 1, 2 and 5.

Results: The effect of all transmembrane compounds and nucleotide derivatives tested in activating T1R1/T1R3 is shown in table 26. Treatment of HEK293 cells expressing T1R1/T1R3 receptors with several of the transmembrane compounds alone in buffer resulted in activation of T1R1/T1R3 as indicated by the change in intracellular calcium levels (ΔF/F₀), as shown in Table 24A-O. When these compounds were combined with 0.2 mM IMP, or a mixture of 0.2 mM IMP and 20 mM alanine, the transmembrane compounds were more effective in activating T1R1/T1R3, as evidenced by a decrease in the EC50 concentration of the transmembrane compounds (Table 24A-O and FIGS. 58-75). Table 24A-O show the EC50 for each of the active transmembrane compound alone (in buffer) and in the presence of L-alanine, IMP, and L-alanine+IMP. Table 25 and FIG. 71 show the EC50 and dose response curves for the control compounds (20 mM of an amino acid in the presence of 0.2 mM IMP). Without being bound to a particular theory, these results show that nucleotides alone, e.g., IMP, or combinations of nucleotides and amino acids, e.g., IMP and alanine, function as positive modulators of the disclosed transmembrane compounds, thereby resulting in the reduction in the amount of the transmembrane compound necessary to sufficiently activate the T1R1/T1R3 receptor.

Tables 24A-O. EC₅₀ of transmembrane compounds in activating 1R1/T1R3.

TABLE 24A 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione T1R1/T1R3 EC₅₀ alone in buffer 0.26 ± 0.01 mM +20 mM L-alanine 0.31 ± 0.05 mM +0.2 mM IMP 0.52 ± 0.19 mM +20 mM L-alanine + 0.2 mM IMP >0.3 mM

TABLE 24B 4-benzyl-3-butyl-1-(2-oxo-2-(pyrrolidin-1-yl)ethyl)- 1H-1,2,4-triazol-5(4H)-one T1R1/T1R3 EC₅₀ alone in buffer >0.6 mM +20 mM L-alanine >0.6 mM +0.2 mM IMP >0.3 mM +20 mM L-alanine + 0.2 mM IMP >0.3 mM

TABLE 24C 2-((3,5-dichlorophenyl)carbamoyl)cyclo- hexanecarboxylic acid - T1R1/T1R3 1-yl)ethyl)-1H-1,2,4-triazol-5(4H)-one EC₅₀ alone in buffer 0.37 ± 0.05 mM +20 mM L-alanine 0.49 ± 0.01 mM +0.2 mM IMP 0.18 ± 0.01 mM +20 mM L-alanine + 0.2 mM IMP 0.15 ± 0.01 mM

TABLE 24D 4-acetamido-N-(1-(2-hydroxyethyl)-3-phenyl-1H- T1R1/T1R3 pyrazol-5-yl)benzamide EC₅₀ alone in buffer N/A +20 mM L-alanine N/A +0.2 mM IMP >0.1 mM +20 mM L-alanine + 0.2 mM IMP >0.1 mM

TABLE 24E T1R1/T1R3 (Diphenylacetyl)-carbamic acid ethyl ester EC₅₀ alone in buffer 0.25 ± 0.01 mM +20 mM L-alanine 0.38 ± 0.01 mM +0.2 mM IMP 0.12 ± 0.02 mM +20 mM L-alanine + 0.2 mM IMP 0.10 ± 0.01 mM

TABLE 24F T1R1/T1R3 N,N′-(butane-1,4-diyl)dinicotinamide EC₅₀ alone in buffer N/A +20 mM L-alanine N/A +0.2 mM IMP 0.60 ± 0.09 mM +20 mM L-alanine + 0.2 mM IMP 0.61 ± 0.36 mM

TABLE 24G T1R1/T1R3 N-phenethylnicotinamide EC₅₀ alone in buffer 0.37 ± 0.02 mM +20 mM L-alanine >0.3 mM +0.2 mM IMP 0.35 ± 0.1 mM +20 mM L-alanine + 0.2 mM IMP 0.22 ± 0.02 mM

TABLE 24H T1R1/T1R3 2-amino-N-phenethylbenzamide EC₅₀ alone in buffer 0.42 ± 0.01 mM +20 mM L-alanine 0.48 ± 0.01 mM +0.2 mM IMP 0.14 ± 0.02 mM +20 mM L-alanine + 0.2 mM IMP 0.09 ± 0.01 mM

TABLE 24I T1R1/T1R3 N-phenethylbenzo[d][1,3]dioxole-5-carboxamide EC₅₀ alone in buffer 0.12 ± 0.07 mM +20 mM L-alanine >1.0 mM +0.2 mM IMP 0.04 ± 0.01 mM +20 mM L-alanine + 0.2 mM IMP 0.03 ± 0.01 mM

TABLE 24J T1R1/T1R3 N-phenethylbenzamide EC₅₀ alone in buffer 0.33 ± 0.01 mM +20 mM L-alanine >1.0 mM +0.2 mM IMP 0.12 ± 0.03 mM +20 mM L-alanine + 0.2 mM IMP 0.09 ± 0.02 mM

TABLE 24K T1R1/T1R3 N-Benzoyl-DL-Leucinamide EC₅₀ alone in buffer >0.6 mM +20 mM L-alanine >1.0 mM +0.2 mM IMP 0.47 ± 0.07 mM +20 mM L-alanine + 0.2 mM IMP 0.91 ± 0.23 mM

TABLE 24L T1R1/T1R3 N-(2-(1H-Indol-3-yl)ethyl)nicotinamide EC₅₀ alone in buffer 0.15 ± 0.03 mM +20 mM L-alanine >0.1 mM +0.2 mM IMP 0.05 ± 0.01 mM +20 mM L-alanine + 0.2 mM IMP 0.04 ± 0.10 mM

TABLE 24M T1R1/T1R3 N-Benzyl-L-phenylalanine methyl ester hydrochloride EC₅₀ alone in buffer 0.03 ± 0.002 mM +20 mM L-alanine 0.05 ± 0.001 mM +0.2 mM IMP 0.02 ± 0.001 mM +20 mM L-alanine + 0.2 mM IMP 0.02 ± 0.002 mM

TABLE 24N T1R1/T1R3 6-Thioguanosine-5′-O-diphosphate (6-T-GDP) EC₅₀ alone in buffer N/A +20 mM L-alanine 0.18 ± 0.02 mM +0.2 mM IMP N/A +20 mM L-alanine + 0.2 mM IMP 0.28 ± 0.05 mM

TABLE 24O 6-Chloropurine riboside-5′-O-triphosphate (6-Cl- T1R1/T1R3 PuTP) EC₅₀ alone in buffer >0.03 mM +20 mM L-alanine >0.03 mM +0.2 mM IMP >0.03 mM +20 mM L-alanine + 0.2 mM IMP >0.03 mM

TABLE 25 Positive and negative controls for transmembrane compound activation of T1R1/T1R3 EC₅₀ (effect with Type of control Compound Name 0.2 mM IMP) Positive L-Alanine 12.3 ± 1.3 mM L-Serine 15.1 ± 2.3 mM L-Cysteine 43.9 ± 5.6 mM L-Phenylalanine 0.5 ± 0.1 mM L-Tryptophan 0.5 ± 0.1 mM L-Histidine 15.3 ± 3.2 mM L-Methionine 33.9 ± 1.5 mM L-Tyrosine 9.3 ± 1.7 mM IMP 0.21 ± 0.04 mM GMP 0.09 ± 0.02 mM Negative L-Proline — MSG — Quinine HCL — D-Glucose —

TABLE 26 Modulation of T1R1/T1R3 by transmembrane compounds +Alanine (20 mM) + +buffer +Alanine (20 mM) +IMP (0.2 mM) IMP (alone) Max Max Max Max (0.2 mM) EC₅₀ EC₅₀ resp resp % Thres EC₅₀ resp resp % Thres EC₅₀ Compound Name Activity (mM) (mM) ΔF/F of IMP (mM) (mM) ΔF/F of Ala (mM) (mM) L-Cysteine N/T N/D N/D N/D N/D N/D 43.9 0.5800 74.6 10.0 N/D L-Phenylalanine N/T N/D N/D N/D N/D N/D 0.5 0.5900 75.9 0.1 N/D L-Tryptophan N/T N/D N/D N/D N/D N/D 0.5 0.6750 86.8 0.1 N/D L-Serine N/T N/D N/D N/D N/D N/D 15.1 0.7775 100.0 3.0 N/D L-Histidine N/T N/D N/D N/D N/D N/D 15.3 0.2825 36.3 10.0 N/D L-Alanine N/T N/D N/D N/D N/D N/D 12.3 0.7775 100.0 3.0 N/D L-Methionine N/T N/D N/D N/D N/D N/D 33.9 0.3500 45.0 30.0 N/D Inosine monophosphate (IMP) N/T N/D 0.21 0.6850 100.0 0.03 N/D N/D N/D N/D N/D Guanosine monophosphate N/T N/D 0.09 0.7850 114.6 0.01 N/D N/D N/D N/D N/D (GMP) L-Proline N/T N/D N/D N/D N/D N/D CNBD CNBD CNBD CNBD N/D MSG (Glutamic acid sodium salt) N/T N/D N/D N/D N/D N/D CNBD CNBD CNBD CNBD N/D Quinine HCL N/T CNBD N/D N/D N/D N/D N/D N/D N/D N/D N/D D-Glucose N/T CNBD N/D N/D N/D N/D N/D N/D N/D N/D N/D L-Tyrosine N/T N/D N/D N/D N/D N/D 9.3 0.2958 38.0 3.0 N/D Mars1: 1-benzyl-3-(2-oxo-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenylethyl)imidazolidine-2,4,5- trione N-(4-(2-(3-benzyl-2,4,5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D trioxoimidazolidin-1- yl)acetyl)phenyl)methanesulfonamide 1-benzyl-3-(2-(2,3-dihydro-1H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D inden-5-yl)-2- oxoethyl)imidazolidine-2,4,5- trione N-(4-(2-(3-(furan-2-ylmethyl)- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2,4,5-trioxoimidazolidin-1- yl)acetyl)phenyl)acetamide 1-benzyl-3-(2-(4-bromophenyl)- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2-oxoethyl)imidazolidine-2,4,5- trione 1-benzyl-3-(2-(4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (methylsulfonyl)phenyl)-2- oxoethyl)imidazolidine-2,4,5- trione 1-(furan-2-ylmethyl)-3-(2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D mesityl-2- oxoethyl)imidazolidine-2,4,5- trione 1-benzyl-3-(2-(2,3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D dihydrobenzo[b][1,4]dioxin-6- yl)-2-oxoethyl)imidazolidine- 2,4,5-trione 1-benzyl-3-(2-(2,4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D dimethoxyphenyl)-2- oxoethyl)imidazolidine-2,4,5- trione 1-(2-(3-methoxyphenyl)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D oxoethyl)-3-(thiophen-2- ylmethyl)imidazolidine-2,4,5- trione 1-(2-(3,4-dimethylphenyl)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D oxoethyl)-3-(2-(thiophen-2- yl)ethyl)imidazolidine-2,4,5- trione 1-(2-oxo-2-phenylethyl)-3-(2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (thiophen-2- yl)ethyl)imidazolidine-2,4,5- trione 1-(2-(benzo[d][1,3]dioxol-5-yl)- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2-oxoethyl)-3-(2-(thiophen-2- yl)ethyl)imidazolidine-2,4,5- trione 1-benzyl-3-(2-(3,4-dihydro-2H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D benzo[b][1,4]dioxepin-7-yl)-2- oxoethyl)imidazolidine-2,4,5- trione 2-(3-benzyl-2,4,5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D trioxoimidazolidin-1-yl)-N-(3- cyanophenyl)acetamide N-benzyl-2-(3-benzyl-2,4,5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D trioxoimidazolidin-1- yl)acetamide N-(3-chloro-4-methoxyphenyl)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (3-(4-methoxyphenyl)-2,4,5- trioxoimidazolidin-1- yl)acetamide 1-(cyclohexylmethyl)-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D cyclopentylimidazolidine-2,4,5- trione 2-(3-benzyl-2,4,5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D trioxoimidazolidin-1-yl)-N- methyl-N-(4- methylcyclohexyl)acetamide 1-(2-oxo-2-(3-oxo-3,4-dihydro- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2H-benzo[b][1,4]oxazin-6- yl)ethyl)-3-(2-(thiophen-2- yl)ethyl)imidazolidine-2,4,5- trione 1-(2-(2-methoxyphenyl)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D oxoethyl)-3-(thiophen-2- ylmethyl)imidazolidine-2,4,5- trione 1-(2-(naphthalen-1-yl)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D oxoethyl)-3-(2-(thiophen-2- yl)ethyl)imidazolidine-2,4,5- trione 1-benzyl-3-(2-(5-chlorothiophen- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2-yl)-2-oxoethyl)imidazolidine- 2,4,5-trione 1-(2-(benzofuran-2-yl)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D oxoethyl)-3-benzylimidazolidine- 2,4,5-trione methyl 4-((3-benzyl-2,4,5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D trioxoimidazolidin-1- yl)methyl)benzoate N-(4-(2-(3-benzyl-2,4,5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D trioxoimidazolidin-1- yl)acetyl)phenyl)propionamide 1-benzyl-3-(2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D chlorobenzyl)imidazolidine-2,4,5- trione 1-benzyl-3-(3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenylpropyl)imidazolidine- 2,4,5-trione 1,3-dibenzyl-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D thioxoimidazolidine-4,5-dione 1,3-dibenzylpyrimidine- active 0.26 0.31 0.2925 42.7 0.1 0.52 0.2775 35.7 0.1 >0.3 2,4,6(1H,3H,5H)-trione 1-benzyl-5-(4-hydroxy-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methoxybenzyl)pyrimidine- 2,4,6(1H,3H,5H)-trione 1-benzyl-3-phenyltetrahydro-1H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D thieno[3,4-d]imidazol-2(3H)-one 5,5-dioxide 2-(1-benzyl-2-oxo-1H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D imidazo[4,5-c]pyridin-3(2H)-yl)- N-isobutylacetamide 2-(4-benzyl-3-methyl-5-oxo-4,5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D dihydro-1H-1,2,4-triazol-1-yl)-N- (1-(thiophen-2-yl)ethyl)acetamide 4-benzyl-3-butyl-1-(2-oxo-2- active >0.6 >0.6 0.8900 129.9 0.3 >0.6 1.5025 193.2 0.1 >0.6 (pyrrolidin-1-yl)ethyl)-1H-1,2,4- triazol-5(4H)-one 1-(2-(4-fluorophenyl)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D oxoethyl)-4-(4- methylbenzyl)pyrazine- 2,3(1H,4H)-dione 1,3-diphenethylpyrimidine- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2,4,6(1H,3H,5H)-trione 3-(4-methylbenzyl)-1-(2-(p- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D tolyloxy)ethyl)pyrimidine- 2,4(1H,3H)-dione N-(3-chloro-4-methoxyphenyl)-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (5-(4-chlorobenzyl)-1,1-dioxido- 1,2,5-thiadiazolidin-2- yl)acetamide 4,4′-((2,2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D dioxidobenzo[c][1,2,5]thiadiazole- 1,3- diyl)bis(methylene))dibenzamide 3,3′-((2,2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D dioxidobenzo[c][1,2,5]thiadiazole- 1,3- diyl)bis(methylene))dibenzonitrile 2,5-diphenethyl-1,3,4-thiadiazole N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N-(4-methyl-5-(pyridin-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D yl)thiazol-2-yl)nicotinamide 5-(3-chlorobenzyl)-N-(pyridin-4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D ylmethyl)-1,3,4-oxadiazole-2- carboxamide N,N′-(4H-1,2,4-triazole-3,5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D diyl)bis(4-fluorobenzamide) N-(2-chlorobenzyl)-5-(2,4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D dichlorobenzyl)-1,3,4-oxadiazole- 2-carboxamide 1-benzyl-3-(benzylamino)-1H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D pyrrole-2,5-dione 1-benzyl-3-(benzylamino)-1H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D pyrrole-2,5-dione 2-((3-(4-acetylphenyl)-4-methyl- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 2-oxo-2,3-dihydro-1H-imidazol- 1-yl)methyl)-5-fluorobenzonitrile 6-methoxy-N-(2-(1-(4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methylbenzyl)-2-oxo-1H- imidazo[4,5-b]pyridin-3(2H)- yl)ethyl)nicotinamide N-(2-(1-benzyl-2-oxo-1H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D imidazo[4,5-b]pyridin-3(2H)- yl)ethyl)-3-methoxybenzamide 1,3-dibenzyltetrahydro-1H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D thieno[3,4-d]imidazol-2(3H)-one 5,5-dioxide 1-(4-chlorobenzyl)-3-(2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenoxyethyl)-1H- benzo[d]imidazol-2(3H)-imine 2-(3-benzyl-2-imino-2,3-dihydro- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 1H-benzo[d]imidazol-1-yl)-1- (3,4-dichlorophenyl)ethanone 1-(2,6-dihydroxy-4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methoxyphenyl)-3-phenylpropan- 1-one 3-(3,4-dimethoxyphenyl)-1-(2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D hydroxy-4,6- dimethoxyphenyl)propan-1-one (E)-1-(2-hydroxyphenyl)-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenylprop-2-en-1-one (E)-1-(4-methoxyphenyl)-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenylprop-2-en-1-one (E)-3-(3,4-dimethoxyphenyl)-1- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (2,4,6-trimethoxyphenyl)prop-2- en-1-one (E)-1-(4-hydroxy-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methoxyphenyl)-3-(4- hydroxyphenyl)prop-2-en-1-one 2-(2-aminobenzamido)propanoic N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D acid methyl 2-benzamido-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenylpropanoate methyl 2-(2,3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D dihydroxybenzamido)-3- hydroxypropanoate N-(5-methylthiazol-2-yl)-9H- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D xanthene-9-carboxamide N-(5-acetyl-4-methylthiazol-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D yl)-2,2-diphenylacetamide N-(4-methylthiazol-2-yl)-2,2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D diphenylacetamide N-(2-methyl-3-nitrobenzyl)-N- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (pyridin-3-yl)ethanesulfonamide N-benzyl-2-isopropoxy-N- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (pyridin-4-yl)ethanesulfonamide 5-(phenoxymethyl)-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenyloxazolidin-2-one N-(4-chloro-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D fluorophenyl)quinoline-2- carboxamide N-(4-chlorophenyl)-6-(4H-1,2,4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D triazol-4-yl)picolinamide 2-((3,5- active 0.37 0.49 1.1200 163.5 0.1 0.18 1.2275 157.9 0.03 0.15 dichlorophenyl)carbamoyl) cyclohexanecarboxylic acid 1-(2,4-dimethylphenyl)-4-(3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methylpiperidin-1-yl)-1H- pyrazolo[3,4-d]pyrimidine (1E,2E)-1,2-bis(3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D fluorobenzylidene)hydrazine 2-((E)-((E)- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D benzylidenehydrazono)methyl)phenol 2-((E)-((E)-(4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D bromobenzylidene)hydrazono)methyl)- 4-nitrophenol 3-cyano-N-(3-(2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D methoxyphenyl)-1H-pyrazol-5- yl)benzamide N-(1-(2-hydroxyethyl)-3-phenyl- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 1H-pyrazol-5-yl)-3- methoxybenzamide 4-acetamido-N-(1-(2- active CNBD CNBD 0.0125 1.8 CNBD >1.0 0.4575 58.8 CNBD >1.0 hydroxyethyl)-3-phenyl-1H- pyrazol-5-yl)benzamide 2-(4-fluorophenyl)-1- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D tosylpyrrolidine N-(3-(pyridin-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D ylethynyl)phenyl)propionamide 1-(5-(phenylethynyl)thiophen-2- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D yl)ethanone (E)-1-(5- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (phenylethynyl)thiophen-2- yl)ethanone oxime N-(3-methoxypropyl)-4- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D (phenylethynyl)benzamide Ro 67-4853 N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Ro 01-6128 active 0.25 0.38 0.5125 74.8 0.3 0.12 1.5525 199.7 0.01 0.10 VU 0361737 N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D AMG-073 HCl (Cinacalcet N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D hydrochloride) DCB N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D VU 0357121 N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N,N′-(butane-1,4- active CNBD CNBD 0.0650 9.5 1.0 0.60 0.3825 49.2 0.1 0.61 diyl)dinicotinamide N-phenethylnicotinamide active 0.37 >0.3 0.5725 83.6 0.1 0.35 1.1625 149.5 0.03 0.22 N-(2-(1H-indol-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D yl)ethyl)nicotinamide 2-amino-N-phenethylbenzamide active 0.42 0.48 0.6400 93.4 0.3 0.14 1.5125 194.5 0.01 0.09 N-isobutylbenzo[d][1,3]dioxole- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 5-carboxamide N- active 0.12 CNBD 0.0775 11.3 1.0 0.04 0.7150 92.0 0.01 0.03 phenethylbenzo[d][1,3]dioxole-5- carboxamide N-(1-amino-1-oxo-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenylpropan-2-yl)benzamide N-(1-(methylamino)-1- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D oxopropan-2-yl)cinnamamide N-phenethylbenzamide active 0.33 >1.0 0.1400 20.4 0.6 0.12 0.9800 126.0 0.01 0.09 2-((carboxymethyl)amino)-3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D phenylpropanoic acid N-Benzoyl-DL-Leucinamide active >0.6 >1.0 0.8600 125.5 0.3 0.47 1.5900 204.5 0.1 0.91 N-(2-(1H-Indol-3- active 0.15 >0.1 1.1400 166.4 0.03 0.05 1.3225 170.1 0.003 0.04 yl)ethyl)nicotinamide N-Benzyl-L-phenylalanine active 0.03 0.05 1.8725 273.4 0.01 0.02 2.2500 289.4 0.003 0.02 methyl ester hydrochloride N-benzyl-L-Leucine N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N-(sec-butyl)-2H-1,3- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D benzodioxole-5-carboxamide 2′-/3′-O-(N′- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Methylanthraniloyl)inosine- 5′-O-monophosphate (MANT- 5′-IMP) N6-Cyclopentyladenosine-5′-O- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D diphosphate (6-cPe-ADP/ cpADP) 2′-/3′-O-(N′- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Methylanthraniloyl)adenosine-5′- O-diphosphate (MANT-ADP) 6-Thioguanosine-5′-O- active CNBD 0.18 1.0470 152.8 0.03 CNBD 0.0720 9.3 CNBD 0.28 diphosphate (6-T-GDP) 6-Chloropurine riboside-5′-O- active >0.3 >0.3 0.3450 50.4 0.01 >0.3 0.2350 30.2 0.003 >0.3 triphosphate (6-Cl-PuTP) 2′-/3′-O-(N′- N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Methylanthraniloyl)adenosine- 5′-O-triphosphate (MANT-ATP) benzyl cinnamate (EU Approved) N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Isobutyl benzoate; Flavis 09.705 N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Tartrazine; Yellow No. 5 N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Chromotrope FB; Carmosine N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Food Red 3 Isobutyl phenylacetate Flavis N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D 09.705 (EU Approved) Phenethyl phenylacetate (EU N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Approved) Benzyl benzoate (EU Approved) N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D Benzyl salicylate (EU Approved) N/A N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D = Not determined N/T = Not tested N/A = Not active CNBD = Could not be determined

Example 8—Activation of T1R1/T1R3 Receptor by Transmembrane Compounds

The present example describes the activation of the T1R1/T1R3 receptor by transmembrane compounds in vitro, wherein the transmembrane compounds function as receptor agonists and/or positive allosteric modulators (PAMs). When functioning as PAMs, the transmembrane compounds increase the effect of nucleotides and amino acids on the activity of the receptor.

Based on the in silico modeling described in Example 4, putative T1R1/T1R3 transmembrane compounds were identified and selected for further testing in vitro as receptor agonists and/or PAMs. In vitro functional characterization of the selected compounds was used to evaluate the effectiveness of the putative transmembrane compounds as agonists and/or PAMs in activating the T1R1/T1R3 receptor.

Agonist Screening Method:

HEK293 cells that stably express T1R3 and inducibly express T1R1 were exposed to a transmembrane compound. Activation of the T1R1/T1R3 receptor was detected by a change in intracellular calcium levels using a calcium sensitive fluorescent dye and/or a luminescent reporter system. Cells that do not express the T1R1/T1R3 receptor were used as a control. A FLIPR® Tetra or a FlexStation® 3 was used for data capture.

Each transmembrane compound was tested at concentrations of 0.01 mM, 0.1 mM, and 1 mM. Dose response curves were generated for each transmembrane compound that activated T1R1/T1R3. To generate the dose response curves, the transmembrane compound was tested at concentrations of between 0.0001 mM and 1.0 mM in the presence of GMP and Ala. Dose response curves were created wherein Ala was held constant at 20 mm, and GMP was increased from 0.001 to 1 mM (specifically, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 0.6, and 1 mM). Similarly, GMP was held constant at 1 mM and the concentration of Ala was varied between 0.1 and 100 mM (specifically, 0.1, 0.3, 1, 3, 10, 30, 60, and 100 mM).

PAM Screening Method:

HEK293 cells that stably express T1R3 and inducibly express T1R1 were exposed to transmembrane compound alone or in combination with Ala and GMP to activate the umami receptor. Activation of the T1R1/T1R3 receptor was detected by a change in intracellular calcium levels using a calcium sensitive fluorescent dye and/or a luminescent reporter system. Cells that do not express the T1R1/T1R3 receptor were used as a control. A FLIPR® Tetra or a FlexStation® 3 was used for data capture.

Dose response curves were generated for each transmembrane compound at concentrations of 0 (buffer only), 0.01, 0.1 and 1 mM, wherein each of the three concentrations of transmembrane compound was tested in combination with 0.03 mM GMP+100 mM Ala (T1); 0.6 mM GMP+10 mM Ala (T2); 0.1 mM GMP+60 mM Ala (T3) and 0.3 mM GMP+60 mM Ala (T4). The T4 mixture was also tested in a 2-fold dilution series (the T4(×) Concentration). In these experiments, the first test concentration was 2× (2 times) the T4 concentration. The subsequent test concentrations were 2-fold dilutions from that (1×, 0.5×etc.). This dilution series was tested with or without the addition of the test compound at a constant 0.3 mM concentration.

Activation of the umami receptor was also determined in the presence of 1 mM GMP+100 mM Ala to generate a “maximum” umami receptor activation level. A compound was classed as a PAM if the response to any combination of compound+Ala+GMP was greater than the sum of the response to the compound alone and the response to GMP+Alanine alone.

Results:

As shown in Table 27, 24 different transmembrane compounds were tested, and nine were identified as a T1R1/T1R3 agonist, PAM, or both.

TABLE 27 Transmembrane compounds that function as a T1R1/T1R3 agonist, PAM, or both. T1R1/T1R3 T1R1/T1R3 Transmembrane compound name agonist PAM 1 1-benzyl-3-(2-oxo-2-phenylethyl)imidazolidine-2,4,5- No No trione 2 1-benzyl-3-(2-(2,3-dihydro-1H-inden-5-yl)-2- No No oxoethyl)imidazolidine-2,4,5-trione 3 1-benzyl-3-(2-(4-bromophenyl)-2- No No oxoethyl)imidazolidine-2,4,5-trione 4 1-benzyl-3-(2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2- No No oxoethyl)imidazolidine-2,4,5-trione 5 1-benzyl-3-(2-(2,4-dimethoxyphenyl)-2- No No oxoethyl)imidazolidine-2,4,5-trione 6 1-(2-oxo-2-phenylethyl)-3-(2-(thiophen-2- No No yl)ethyl)imidazolidine-2,4,5-trione 7 1-(2-(benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-(2- No No (thiophen-2-yl)ethyl)imidazolidine-2,4,5-trione 8 1-benzyl-3-(2-(3,4-dihydro-2H-benzo[b][1,4]dioxepin-7- No Weak PAM yl)-2-oxoethyl)imidazolidine-2,4,5-trione 9 2-(3-benzyl-2,4,5-trioxoimidazolidin-1-yl)-N-(3- No No cyanophenyl)acetamide 10 1-(2-(2-methoxyphenyl)-2-oxoethyl)-3-(thiophen-2- No No ylmethyl)imidazolidine-2,4,5-trione 11 1-(2-(naphthalen-1-yl)-2-oxoethyl)-3-(2-(thiophen-2- No No yl)ethyl)imidazolidine-2,4,5-trione 12 1-benzyl-3-(2-(5-chlorothiophen-2-yl)-2- No Weak PAM oxoethyl)imidazolidine-2,4,5-trione 13 1,3-dibenzyl-2-thioxoimidazolidine-4,5-dione No No 14 N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide Yes Yes 15 N-Benzyl-L-phenylalanine methyl ester hydrochloride Yes Yes 16 N-Benzyl-D-Phenylalanine methyl ester hydrochloride Yes Yes 17 N-alpha-Benzyl-N-alpha-methyl-L-phenylalanine methyl No No ester hydrochloride 18 Benzyl-L-leucine methyl ester hydrochloride Yes Yes 19 Methyl-2-benzylamino-2-phenylacetate Yes Yes 20 L-Phenylalanine benzyl ester hydrochloride Yes Yes 21 L-Tyrosine benzyl ester No No 22 N-Benzyl-L-Tyrosine No No 23 2-((4-methylbenzyl)amino)-3-phenylpropanoic acid No No 24 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione Yes No

As described by Table 28, nine of the compounds tested were active as T1R1/T1R3 agonists and/or PAMs. FIGS. 72-80 show the dose response curves for the agonist profiling and PAM profiling for each compound identified as a T1R1/T1R3 agonist and/or PAM.

Without being bound to a particular theory, these results show that transmembrane compounds alone can activate T1R1/T1R3 as agonists, and can also function as positive allosteric modulators of GMP and Ala activation of T1R1/T1R3, thereby reducing the amount of agonist necessary to sufficiently activate the T1R1/T1R3 receptor.

Example 9—Identification of T1R1 Transmembrane Compound Interacting Residues

The present example describes the in silico identification of amino acids within T1R1 that interact with transmembrane compounds that bind to T1R1.

Methods:

The cat T1R1 is a Group C G protein-coupled receptor (GPCR), as are T1R2, T1R3, CaSR, GabaB, and mGlu's. Group C GPCRs comprise (1) a large external domain, called a Venus Flytrap domain (VFT), (2) a 7 transmembrane domain (7TM), and (3) a cysteine rich domain which connects VFT and 7TM. A homology model of the cat T1R1 7TM domain was constructed based on crystal structures 4OR2 and 4OO9 from the Protein Data Bank (Berman et al., Nucleic Acids Research, 28: 235-242 (2000)). 4OR2 and 4OO9 are crystal structures of parts of two metabotropic glutamate receptors that are Group C GPCRs. 4OR2 is the crystal structure of the transmembrane domain of mGluR1 with a bound negative allosteric modulator (NAM) (Wu et al., Science. 2014 Apr. 4; 344(6179):58-64. Epub 2014 Mar. 6.). 4OO9 is the crystal structure of the transmembrane domain of mGluR5 with a bound NAM (Dore et al., Nature. 2014 Jul. 31; 511(7511):557-62. Epub 2014 Jul. 6). The models were built using the I-TASSER Suite of programs (Yang et al., Nature Methods, 12: 7-8 (2015)) and the Modeller software package (Eswar et al., Curr Protoc Bioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6.30 (2006)), which is part of the DiscoveryStudio (DS) suite of programs from Dassault Systemes, BIOVIA Corp., San Diego, Calif., USA. There is approximately 25% sequence identity between mGluR1 and cat T1R1 7TM domains. N-Benzyl-L-phenylalanine methyl ester was docked into an allosteric site of the cat T1R1 7TM in the T1R1 model using the docking program BioDock from BioPredict, Inc. (Oradell, N.J., USA). For docking amino acids and nucleotides to the VFT domain, a similar protocol was used to that described for modeling nucleotide docking with the VFT domain.

Results:

In the docking model, N-Benzyl-L-phenylalanine methyl ester interacts with the following amino acids of the allosteric 7TM binding site of cat T1R1: Ala795, Ala796, and Asn792, which are on helix 7 of the 7TM; Trp773 and Phe776, which are on helix 6 of the 7TM; Ala731, Phe728, Leu730, Phe732, and Asn735, which are on helix 5 of the 7TM; Ala689, Ser686, Gln690, Ile693, Cys694 and Leu695, which are on helix 4 of the 7TM; and Arg634, Gln635, Phe642, Ala639, Ala643, and Leu638, which are on helix 3 of the 7TM (FIG. 28. The ester group of N-Benzyl-L-phenylalanine methyl ester forms a hydrogen bond to Asn735. Models of other transmembrane compounds illustrate that the ligand may form hydrogen bonding interactions to Asn735, Ser686, or both.

The majority of the hydrophobic interactions between N-Benzyl-L-phenylalanine methyl ester and the 7TM domain of T1R1 occur between the ligand and Trp773, Phe776, Phe732, Phe728, Leu730, Leu695, Leu638, and Phe642. These amino acids also provide for the majority of the hydrophobic interactions between other T1R1 transmembrane ligands and the T1R1 transmembrane domain.

A striking feature of the T1R1 active site is the number of residues that can undergo ring stacking interactions to a bound ligand. The model of N-Benzyl-L-phenylalanine methyl ester bound to T1R1 7TM shows ring-stacking interactions from the benzyl group to the transmembrane domain. This feature is common to models of other active transmembrane compounds bound to T1R1 7TM. These may contribute both to binding and to stabilization of the T1R1/T1R3 active conformation. T1R1 7TM amino acids that can form such interactions include Trp773, Phe776, Phe732, Phe728, and Phe642.

Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Patents, patent applications, publications, product descriptions and protocols are cited throughout this application the disclosures of which are incorporated herein by reference in their entireties for all purposes. 

1. A flavor composition comprising a compound of Formula Nt-1 comprising the structure

wherein R₁ is selected from the group consisting of a substituted or unsubstituted purine or a substituted or unsubstituted pyrimidine; wherein R₂ is selected from monophosphate, diphosphate, triphosphate, —OP(W)(OH)₂, —OP(W)(OH)OP(W)(OH)₂, —OP(W)(OH)OP(W)(OH)OP(W)(OH)₂, —OS(O)₂aryl(H), —OS(O)₂aryl(CH₃), —P(W)(OH)₂, —OP(W)(OH)OS(O)₂(OH), —OP(W)(OH)Z, —P(W)(OH)OP(W)(OH)₂, —O(CH₂)₁₋₄OP(W)(OH)₂, —OS(W)(OH)₂, —OP(W)(OH) CH₂OP(W)(OH)₂, —OP(W)(OH)OP(W)O(CH₂)₁₋₄R₁₈, —(CH₂)₀₋₄COOH, —(CH₂)₀₋₄S(O)(OH)₂, —(CH₂)₀₋₄C(O)NHOH and —(CH₂)₀₋₄B(OH)₂; wherein X is selected from O, S, N(R₃) and CH₂; wherein W is selected from O and S; wherein R₃ is selected from H and CH₃; wherein R₄, R₅, R₁₄, R₁₅, R₁₈ are independently selected from H, OH, SH, CH₂, CH₃, OR₆, SR₆, CH₂CH₃, lower alkyl branched and unbranched (C₁-C₆), XC(O)lower alkyl, —XC(O)CH₂Ph, —P(W)(OH)₂, —XC(O)PhR₁₁, —OP(O)(OH)O, OCH₃, N(R₁₁, R₁₇), —O(C)n R₁₁, R₁₇O—, N(H or independently lower alkyl)₂₃, COOR₁₁, —OC(W)NH(CH₂)₁₋₆NH₂, —OC(W)NH(CH₂)₁₋₆R₄; wherein Z is selected from piperadine, morpholine, piperizine, N-methyl-piperizine, N(R₁₆)(R₁₇); wherein R₆, R₁₁, R₁₆ and R₁₇ are selected independently from H, OH, SH, CH₂, CH₃, OCH₃, COOR, N(R₁₂)(R₁₃), CH₂CH₃, lower alkyl branched and unbranched (C₁-C₆), XC(O)lower alkyl, —XC(O)CH₂Ph, —P(W)(OH)₂, —XC(O)PhR₁₂, —OP(O)(OH)O, OCH₃, N(R₁₂, R₁₃), —O(C)n R₁₂, R₁₃O—, N(H or independently lower alkyl)₂₃ and COOR₁₂; and wherein R₁₂ and R₁₃ are selected independently from H, OH, SH, CH₂, CH₃, OCH₃ and CH_(2C)H₃.
 2. The flavor composition of claim 1, wherein R₁ of Formula Nt-1 is selected from the group consisting of


3. The flavor composition of claim 1, wherein the compound is selected from the group consisting of

and combinations thereof.
 4. The flavor composition of claim 1, further comprising a compound selected from the group consisting of

and a combination thereof.
 5. The flavor composition of claim 1, further comprising a compound selected from the group consisting of

and combinations thereof.
 6. The flavor composition of claim 1, further comprising a compound having the structure


7. The flavor composition of claim 1, further comprising a compound having the structure


8. The flavor composition of claim 1, further comprising a compound selected from the group consisting of

and combinations thereof.
 9. The flavor composition of claim 1, further comprising a compound having the structure


10. The flavor composition of claim 1, further comprising a compound selected from the group consisting of

and combinations thereof.
 11. The flavor composition of claim 1, further comprising a compound having the structure


12. The flavor composition of claim 1, further comprising a compound having the structure


13. The flavor composition of claim 1, further comprising a compound selected from the group consisting of:


14. The flavor composition of claim 1, further comprising an amino acid selected from the group consisting of tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine, and combinations thereof.
 15. The flavor composition of claim 14, wherein the amino acid is alanine.
 16. The flavor composition of claim 1, further comprising an amino acid selected from the group consisting of asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxyl proline, arginine, cystine, glutamine, lysine, valine, ornithine, monosodium glutamate, taurine, and combinations thereof.
 17. The flavor composition of claim 1, further comprising a first amino acid according to claim 14, and a second amino acid according to claim
 16. 18. The flavor composition of claim 1, further comprising a nucleotide selected from the group consisting of guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP, inosine monophosphate (IMP), inosine diphosphate (IDP), inosine triphosphate (ITP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), xanthosine monophosphate (XMP), xanthosine diphosphate (XDP), xanthosine triphosphate (XTP), and combinations thereof.
 19. The flavor composition of claim 18, wherein the nucleotide is selected from the group consisting of guanosine monophosphate (GMP), inosine monophosphate (IMP), and a combination thereof.
 20. The flavor composition of claim 1, further comprising alanine and guanosine monophosphate (GMP).
 21. The flavor composition of claim 1, further comprising glycine and guanosine monophosphate (GMP).
 22. The flavor composition of claim 1, further comprising histidine and guanosine monophosphate (GMP).
 23. The flavor composition of claim 1, further comprising alanine and inosine monophosphate (IMP).
 24. The flavor composition of claim 1, further comprising glycine and inosine monophosphate (IMP).
 25. The flavor composition of claim 1, further comprising histidine and inosine monophosphate (IMP).
 26. The flavor composition of claim 1, further comprising proline and an amino acid selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.
 27. The flavor composition of claim 1, further comprising threonine and an amino acid selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.
 28. The flavor composition of claim 1, further comprising glutamic acid and an amino acid selected from the group consisting of histidine, alanine, glycine, phenylalanine, tryptophan, and tyrosine.
 29. A food product comprising the flavor composition of claim 1, wherein the flavor composition is present at a concentration of from about 0.0001 weight % to about 10 weight % of the food product. 30-32. (canceled)
 33. A method of increasing an umami taste intensity in a food product comprising admixing a food product with the flavor composition of claim 1, wherein the flavor composition is present at a concentration of from about 0.0001 weight % to about 10 weight % of the admixture. 34-35. (canceled)
 36. The method of claim 33, wherein the increase in umami taste intensity comprises an increase in umami aftertaste.
 37. A method of preparing a food product comprising a flavor composition according to claim 1, wherein the method comprises thermal processing of a food product precursor, wherein the flavor composition is generated during the thermal processing. 38-46. (canceled) 